Patent Application
20150150960
The invention relates to Dengue virus proteins, subsequences and portions thereof, including DENV epitopes and modifcations of DENV proteins, subsequences and portions thereof, and uses and methods for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection.
Dengue virus (DENV, or DV) is a mosquito-borne RNA virus in the Flaviviridae family, which also includes West Nile Virus (WNV), Yellow Fever Virus (YFV), and Japanese Encephalitis Virus (JEV). The four serotypes of DENV (DENV1-4) share approximately 65-75% homology at the amino acid level (Fu, et al. Virology 188:953 (1992)). Infections with DENV can be asymptomatic, or cause disease ranging from dengue fever (DF) to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (WHO, Dengue: Guidelines for diagnosis, treatment, prevention and control (2009)). DF is a self-limiting illness with symptoms that include fever, headache, myalgia, retro-orbital pain, nausea, and vomiting. DHF and DSS are characterized by increased vascular permeability, thrombocytopenia, hemorrhagic manifestations, and in the case of DSS, shock, which can be fatal. The incidence of DENV infections has increased 30-fold in the past 50 years (WHO, Dengue: Guidelines for diagnosis, treatment, prevention and control (2009)). DF and DHF DSS are a significant cause of morbidity and mortality worldwide, and therefore a DENV vaccine is a global public health priority. However, vaccine development has been challenging, as a vaccine should protect against all four DENV serotypes (Whitehead, et al. Nat Rev Microbiol 5:518 (2007)).
Severe dengue disease (DHF/DSS) most often occurs in individuals experiencing a secondary infection with a heterologous DENV serotype, suggesting the immune response contributes to the pathogenesis (Sangkawibha, et al. Am J Epidemiol 120:653 (1984); Guzman, et al. Am J Epidemiol 152:793 (1997)). To explain the occurrence of DHF/DSS with secondary infection, two dominant hypotheses: (i) antibody (Ab) dependent enhancement of infection (ADE) and (ii) original T cell antigenic sin have been postulated. Under the ADE hypothesis, serotype cross-reactive antibodies enhance infection of FcγR+ cells during a secondary infection resulting in higher viral loads and more severe disease via a phenomenon known as antibody-dependent enhancement (ADE) (Morens, et al. Clin Infect Dis 19:500 (1994); Halstead, Adv Virus Res 60:421 (2003)). Recent studies have demonstrated DENV-specific antibody can enhance disease in mice (Zellweger, et al. Cell Host Microbe 7: 128 (2010); Balsitis, et al. PLoS Pathog 6:e1000790 (2010)). Under the original T cell antigenic sin hypothesis, it is proposed that serotype cross-reactive memory T cells may respond sub-optimally during secondary infection and contribute to the pathogenesis (Mathew, et al. Immunol Rev 225:300 (2008)). Accordingly, studies have shown serotype cross-reactive T cells can exhibit an altered phenotype in terms of cytokine production and degranulation (Mangada, et al. J Immunol 175:2676 (2005); Mongkolsapaya, et al. Nat Med 9:921 (2003); Mongkolsapaya, et al. J Immunol 176:3821 (2006)). However, another study found the breadth and magnitude of the T cell response during secondary DENV infection was not significantly associated with disease severity (Simmons, et al. J Virol 79:5665 (2005)).
CD4+ T cells can contribute to the host response to pathogens in a variety of ways. They produce cytokines and can mediate cytotoxicity. They also help B cell responses by inducing immunoglobulin class switch recombination (CSR), and help prime the CD8+ T cell response. CD4+ T cells can help the CD8+ T cell response indirectly by activating APCs, for example via CD40L/CD40 (Bevan, Nat Rev Immunol 4:595 (2004)). CD40L on CD4+ T cells is important in activating B cells as well (Elgueta, et al. Immunol Rev 229:152 (2009)). CD4+ T cells can also induce chemokine production that attracts CD8+ T cells to sites of infection (Nakanishi, et al. Nature 462:510 (2009)). However, the requirement for CD4+ T cell help for antibody and CD8+ T cell responses is not absolute, and may be specific to the pathogen and/or experimental system. For instance, it has been shown that CSR can occur in the absence of CD4+ T cells (Stavnezer, et al. Annu Rev Immunol 26:261 (2008)), and the primary CD8+ T cell response is CD4-independent under inflammatory conditions (Bevan, Nat Rev Immunol 4:595 (2004)). This suspected dual role of T cells in protection and pathogenesis is difficult to study in humans, since in most donor cohorts the time point and in case of secondary infections the sequence of infection is unknown, and does not allow direct correlations with T cell responses.
Although many studies have investigated the role of T cells in DENV pathogenesis, the role of T cells in protection versus pathogenesis during DENV infections was, prior to the disclosure herein,unknown. In this regard, the lack of an adequate animal model made such studies impossible, as mice are resistant to infection with this human pathogen (Yauch, et al. Antiviral Res 80:87 (2008)). A mouse model, which allows investigation of adaptive immune responses restricted by human histocompatibility complex (MHC) molecules to DENV infection, would shed light on the role of T cells in protection and/or pathogenesis.
Mice transgenic for human leukocyte antigens (HLA) are widely used to study T cell responses restricted by human MHC molecules and studies in other viral systems have shown the valuable impact of HLA transgenic mice in epitope identification (Kotturi, et al. Immunome Res 6:4 (2010); Kotturi, et al. Immunome Res 5:3 (2009); Pasquetto, et al. J Immunol 175:5504 (2005)). Recently, a mouse-passaged DENV2 strain, S221, which does not replicate to detectable levels in wild-type C57BL/6 mice, was reported to replicate in IFN-α/R‘′″ mice (Yauch, et al. J Immunol 182:4865 (2009)). Using S221 and IFN-αβR−/− mice, a protective role for CD8+ T cells in the response to primary DENV2 infection was reported (Yauch, et al. J Immunol 182:4865 (2009)). The DENV field has been focusing vaccine development efforts towards induction of humoral immunity, because as with other viral vaccines, DENV-specific antibodies (Abs) are assumed to provide the key means of protection against natural infection. However, epidemiologic studies have shown that severe dengue disease is preferentially associated with secondary infections in humans and infants born to DENV-immune mothers. Moreover, recent studies using mouse models have shown DENV-specific Abs can contribute to pathogenesis by mediating antibody-dependent enhancement of infection (ADE). ADE has been demonstrated to enhance viremia and severity of dengue disease in non-human primate (Goncalvez, et al. Proc Natl Acad Sci U S A 104:9422-9427 (2007); Halstead J Infect Dis 140:527-533 (1979); Halstead, et al. J Infect Dis 128:15-22 (1973)) and mouse (Balsitis, et al. PLoS Pathog 6:e1000790 (2010); Zellweger, et al. Cell Host Microbe 7:128-139 (2010)) models, respectively. Despite the potential for ADE, based on a vast number of publications on antibody-mediated protection (reviewed in Innis CAB International, Wallingford, Oxon, UK; New York (1997); Murphy, et al. Annual Rev. of Immunol. 29:587-619 (2011)), the consensus in the field is that induction of protective levels of neutralizing Abs should be the primary objective of dengue vaccination.
Direct evidence linking T cells to increased viremia or pathology has never been shown, although numerous studies have examined T cell responses in the context of Dengue virus (DENV) pathogenesis. Although limited, studies examining T cell-mediated protection against DENV (Calvert, et al. Journal General Virol. 87:339-346 (2006); Kyle, et al. Virology 380:296-303 (2008)) generally assume that T cells play at most a secondary role in protection against DENV reinfection.
The invention is based, in part, on the discovery that DENV vaccine-induced antibody response can mediate ADE and enhance (worsen) DENV disease severity. The invention is also based, in part, on the discovery that CD8+ T cell responses dictate the extent of dengue vaccine-mediated protection. The invention is further based, in part, on the discovery that CD8+ T cell responses can provide protection against DENV infection, including protection against heterologous DENV serotypes, even in the presence of enhancing antibodies.
Thus, the invention provides uses, methods and compositions for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject. In one embodiment, a use or method includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to elicit an anti-Dengue virus T cell response in the subject. In particular aspects, a use or method elicits, stimulates, induces, promotes, increases, or enhances an anti-Dengue virus T cell response in a subject without sensitizing the subject to severe dengue disease (e.g., ADE) upon a secondary or subsequent Dengue virus exposure or infection.
In another embodiment, a use or a method of vaccinating a subject against or providing a subject with protection against a Dengue virus infection includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to vaccinate the subject against or protect the subject against the Dengue virus infection. In a particular, aspect, the use or method does not sensitize the subject to severe dengue disease upon a secondary or subsequent Dengue virus exposure or infection.
In a further embodiment, a use or method of treating a subject for a Dengue virus infection includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to treat the subject for the Dengue virus infection. In a particular, aspect, the use or method does not sensitize the subject to severe dengue disease upon a secondary or subsequent Dengue virus exposure or infection.
The invention also provides compositions including an amount of a Dengue virus protein or subsequence or portion or modification thereof. In various embodiments, these compositions are for use in: eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject, optionally without elicting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection or exposure; in providing a subject with protection against a Dengue virus infection or pathology, or one or more physiological disorders, illness, diseases or symptoms caused by or associated with Dengue virus infection or pathology, optionally without elicting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection; in vaccinating a subject against a Dengue virus infection without elicting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection or exposure; and in treating a subject for a Dengue virus infection, optionally without elicting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection or exposure.
In additional particular embodiments, the uses, methods and compositions are useful for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus CD8+ T cell response, optionally without elicting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection or exposure. In certain embodiments , anti-Dengue virus CD8+ T cell response is directed and/or protective against a plurality of different Dengue virus serotypes. In particular embodiments, the anti-Dengue virus CD8+ T cell response is directed and/or protective against at least two Dengue virus serotypes selected from DENV1, DENV2, DENV3 and DENV4.
In different embodiments of the uses, methods and compositions, the protein comprises or consists of a Dengue virus serotype 1, 2, 3 or 4 protein.
In certain embodiments, a Dengue virus protein is a non-structural protein such as, for example, NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5. In other embodiments, a Dengue virus protein is a structural protein such as, for example, Dengue virus envelope (E) protein, membrane (M) protein or core protein.
In uses, methods and compositions of the invention include those that do not substantially sensitize a subject to severe dengue disease (e.g., via ADE), or elicit, induce, stimulate or promote severe dengue disease, upon a secondary or subsequent Dengue virus infection or exposure. In certain embodiments, severe dengue disease is mediated by antibody dependent enhancement (ADE). In certain embodiment, the severe Dengue virus disease comprises antibody-dependent enhancement of infection.
In certain embodiments of the uses, methods and compositions, the protein administered consists of a single Dengue virus serotype. In other embodiments of the uses, methods and compositions, protein administered comprises a plurality of single Dengue virus serotype proteins administered. In still further embodiments of the uses, methods and compositions, protein administered comprises or consists of one or more Dengue virus serotype 1, 2, 3 or 4 proteins. In particular different embodiments of the uses, methods and compositions, protein administered comprises or consists of one or more Dengue virus serotype 1 proteins, and not a Dengue virus serotype 2, 3 or 4 protein; protein administered comprises or consists of one or more Dengue virus serotype 2 proteins, and not a Dengue virus serotype 1, 3 or 4 protein; protein administered comprises or consists of one or more Dengue virus serotype 3 proteins, and not a Dengue virus serotype 1, 2 or 4 protein; or protein administered comprises or consists of one or more Dengue virus serotype 4 proteins, and not a Dengue virus serotype 1, 2 or 3 protein.
In certain embodiments of the uses, methods and compositions, administration of a protein of a first Dengue virus serotype is effective to vaccinate or provide the subject with protection against one or more Dengue virus serotypes distinct from the first Dengue virus serotype. In particular different embodiments of the uses, methods and compositions, administration of a Dengue virus serotype 1 protein is effective to vaccinate or provide the subject with protection against one or more of Dengue virus serotypes 2, 3 or 4; administration of a Dengue virus serotype 2 protein is effective to vaccinate or provide the subject with protection against one or more of Dengue virus serotypes 1, 3 or 4; administration of a Dengue virus serotype 3 protein is effective to vaccinate or provide the subject with protection against one or more of Dengue virus serotypes 1, 2 or 4; or administration of a Dengue virus serotype 4 protein is effective to vaccinate or provide the subject with protection against one or more of Dengue virus serotypes 1, 2 or 3.
In other embodiments of the uses, methods and compositions, administration of a protein of a first Dengue virus serotype is effective to treat the subject for infection with one or more Dengue virus serotypes distinct from the first Dengue virus serotype. In particular different embodiments of the uses, methods and compositions, administration of a Dengue virus serotype 1 protein is effective to treat the subject for infection with one or more of Dengue virus serotypes 2, 3 or 4; administration of a Dengue virus serotype 2 protein is effective to treat the subject for infection with one or more of Dengue virus serotypes 1, 3 or 4; administration of a Dengue virus serotype 3 protein is effective to treat the subject for infection with one or more of Dengue virus serotypes 1, 2 or 4; or administration of a Dengue virus serotype 4 protein is effective to treat the subject for infection with one or more of Dengue virus serotypes 1, 2 or 3.
In certain embodiments, uses, methods and compositions reduce Dengue virus titer, increasing or stimulating Dengue virus clearance, reduce or inhibit Dengue virus proliferation, reduce or inhibit increases in Dengue virus titer or Dengue virus proliferation, reduce the amount of a Dengue virus protein or the amount of a Dengue virus nucleic acids, or reduce or inhibit synthesis of a Dengue virus protein or a Dengue virus nucleic acid. In other particular embodiments, uses, methods and compositions prevent, reduce, improve or inhibit one or more adverse physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated with Dengue virus infection or pathology. In still further particular embodiments, uses, methods and compositions reduce or inhibit susceptibility to Dengue virus infection or pathology or protect a subject from adverse physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated with an antibody response to a Dengue virus infection.
In other embodiments, invention uses, methods and compositions may be performed or administered prior to exposure to or infection of the subject with the Dengue virus, or substantially contemporaneously with exposure to or infection of the subject with the Dengue virus, or following exposure to or infection of the subject with the Dengue virus. Such exposure or infection includes secondary or subsequent DENV infections (e.g., reinfection).
In further embodiments, invention uses and methods include administering a Dengue virus protein or subsequence or portion or modification thereof in combination with a T-cell stimulatory molecule. In still further embodiments, a composition includes a combination of a Dengue virus protein or portion or modification thereof and a T-cell stimulatory molecule. In particular aspects a T-cell stimulatory molecule is OX40 or CD27.
In particular embodiments of the uses, methods and compositions, the subject is a mammal, for example, a human.
In certain embodiments of the uses, methods and compositions, a subject has not previously been infected with Dengue virus. In other embodiments of the uses, methods and compositions, a subject, prior to administration of the Dengue virus protein, produces antibodies against one or more Dengue virus serotypes. In still further embodiments of the uses, methods and compositions, a subject has previously been infected with Dengue virus.
As disclosed herein, candidate MHC class II (I-Ab)-binding peptides from the entire proteome of DENV2, which is approximately 3390 amino acids and encodes three structural (core (C), envelope (E), and membrane (M)), and seven non-structural (NS) (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) proteins, were identified. Numerous CD4+ T cell and CD8+ T cell epitopes from the structural and non-structural (NS) proteins are also disclosed herein (e.g., Tables 1-4). Immunization with T cell epitopes, such as CD8+ or CD4+ T cell epitopes, before DENV infection resulted in significantly lower viral loads. While CD4+ T cells do not appear to be required for controlling primary DENV infection, immunization contributes to viral clearance.
By way of example, 42 epitopes derived from 9 of the 10 DENV proteins were identified. 80% of the epitopes identified were able to elicit a T cell response in human donors, previously exposed to DENV. The mouse model described herein also reflected response patterns observed in humans. These findings indicate that inducing anti-DENV CD4+ T and/or CD8+ T cell responses by immunization/vaccination will be an effective prophylactic or therapeutic treatment for DENV infection and/or pathology.
In accordance with the invention, there are provided DENV proteins, methods and uses, in which the proteins include or consist of a subsequence, portion, or an amino acid modification of Dengue virus (DV) structural or non-structural (NS) polypeptide sequence from any of DENV serotypes 1, 2, 3 or 4, and the protein elicits, stimulates, induces, promotes, increases, or enhances an anti-DV CD8+ T cell response or an anti-DV CD4+ T cell response. In one embodiment, a protein includes or consists of a subsequence, portion, or an amino acid modification of Dengue virus (DV) structural core (C), membrane (M) or envelope (E) polypeptide sequence, for example, based upon or derived from a DENV1, DENV2, DENV3 or DENV4 serotype. In another embodiment, a protein includes or consists of a subsequence, portion, or an amino acid modification of Dengue virus (DV) NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5 polypeptide sequence, for example, based upon or derived from a DENV1, DENV2, DENV3 or DENV4 serotype.
In particular aspects, a protein includes or consists of a structural or non-structural (NS) polypeptide sequence from a DENV serotype 1, 2, 3 or 4. In additional particular aspects, a protein includes or consists of a sequence set forth in Tables 1-4, or a subsequence thereof or a modification thereof. Exemplary modifications include 1, 2, 3, 4, 5 or 6, 7, 8, 9, 10 or more conservative, non-conservative, or conservative and non-conservative amino acid substitutions.
In certain embodiments, a protein, subsequence, portion, or a modification thereof elicits an anti-DV response. In particular aspects, an anti-DV response includes a CD8+ T cell response and/or a CD4+ T cell response. Such responses can be ascertained, for example, by increased IFN-gamma, TNF-alpha, IL-1alpha, IL-6 or IL-8 production by CD8+ T cells in the presence of the protein; and/or increased CD4+ T cell production of IFN-gamma, TNF, IL-2, or CD40L in the presence of the protein, or killing of peptide-pulsed target cells.
The invention also provides compositions including the proteins, subsequences, portions, or modifications thereof (e.g., T cell epitopes), such as pharmaceutical compositions. Compositions can include one or more proteins, subsequences, portions, or modifications thereof, such as peptides selected from Tables 1-4, or a subsequence or portion thereof, or a modification thereof, as well as optionally adjuvants.
Proteins, subsequences, portions, and modifications thereof (e.g., T cell epitopes) can be used for stimulating, inducing, promoting, increasing, or enhancing an immune response against Dengue virus (DV) in a subject. In one embodiment, a method includes administering to a subject an amount of a DENV protein, subsequence, portion, or a modification thereof sufficient to stimulate, induce, promote, increase, or enhance an immune response against Dengue virus (DV) in the subject, and/or provide the subject with protection against a Dengue virus (DV) infection or pathology, or one or more physiological conditions, disorders, illness, diseases or symptoms caused by or associated with DV infection or pathology.
DENV proteins, subsequences, portions, and modifications thereof (e.g., T cell epitopes) can also be used for treating a subject for a Dengue virus (DV) infection. In one embodiment, a method includes administering to a subject an amount of a DENV protein, subsequence, portion, or a modification thereof sufficient to treat the subject for the Dengue virus (DV) infection.
Exemplary responses, in vitro, ex vivo or in vivo, elicited by proteins, subsequences, portions, or modifications thereof, such as T cell epitopes include, stimulating, inducing, promoting, increasing, or enhancing an anti-DV CD8+ T cell response or an anti-DV CD4+ T cell response. In particular aspects, CD8+ T cells produce IFN-gamma, TNF-alpha, IL-1alpha, IL-6 or IL-8 in response to T cell epitope, and/or CD4+ T cells produce IFN-gamma, TNF, IL-2 or CD40L, or kill peptide-pulsed target cells in response to a T cell epitope. Accordingly, proteins, subsequences, portions, and modifications thereof (e.g., T cell epitopes) can also be used for inducing, increasing, promoting or stimulating anti-Dengue virus (DV) activity of CD8+ T cells or CD4+ T cells in a subject.
In various embodiments, multiple proteins, subsequences, portions, or modifications thereof, for example, multiple Dengue virus (DV) proteins, such as T cell epitopes are employed in the methods and uses of the invention. In particular aspects, a Dengue virus (DV) protein, such as a T cell epitope, includes or consists of one or more sequences set forth in Tables 1-4, or a subsequence or portion thereof, or a modification thereof.
As disclosed herein, T cells contribute towards protection against primary Dengue virus (DENV) infection in clinically relevant mouse models of Dengue virus (Yauch, et al. J Immunol 185:5405-5416 (2010); Yauch, et al. J Immunol 182:4865-4873 (2009)). The studies disclosed herein demonstrate that CD8+ T cells play a critical role in vaccine-mediated protection against DENV infection. Thus, the findings disclosed herein reveal that CD8+ T cell immunity is required for vaccine-mediated protection against DENV, which is contrary to the general consensus in the field that antibodies are essential for immunization or vaccination against Dengue virus.
Furthermore, the studies disclosed herein demonstrate that the responsive CD8+ T cells after administration of a particular DENV serotype can provide the animal with protection against other distinct (heterologous) DENV serotypes. Thus, the studies disclosed herein reveal that a protein or subsequence of a given DENV serotype can be used to provide protection against other distinct DENV serotypes in vaccination and immunization methods and uses. For example, a DENV3 serotype protein or subsequence or portion can be administered to provide a subject with protection against a DENV1, DENV2 and/or DENV4 serotype infection. Moreover, CD8+ T cells that provide protection against distinct DENV serotypes can also provide protection against other distinct DENV serotypes, even in the presence of enhancing antibodies. Thus, the studies disclosed herein also reveal that a protein or subsequence of a given DENV serotype can be used to provide (broad spectrum) protection in subjects who already have developed antibodies against DENV, as a consequence of a prior DENV infection or exposure to DENV (e.g., vaccination or immunization), for example.
In accordance with the invention, there are provide methods and uses for vaccination and immunization to protect against dengue virus infection, and methods and uses for treatment of a Dengue virus infection. Such methods and uses are applicable to providing a subject with protection from Dengue virus infection, and also are applicable to providing treatment to a subject having a Dengue virus infection, particularly subjects that are at risk of severe dengue disease (e.g., ADE mediated DHF or DSS), such as subjects having Dengue virus antibodies, either produced by their own body due to a prior DENV infection or exposure, or through transfer (e.g., maternal transfer or passive immunization or vaccination with against Dengue virus).
In one embodiment, a use or method for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to elicit, stimulate, induce, promote, increase or enhance an anti-Dengue virus T cell response in the subject.
In another embodiment, a use or method for vaccinating or providing a subject with protection against a Dengue virus infection without eliciting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection, includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to vaccinate or provide the subject with protection against the Dengue virus infection.
In another embodiment, a use or method for treating a subject for a Dengue virus infection without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection, includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to treat the subject for the Dengue virus infection.
As used herein, “sensitize” or “sensitizing” refers to causing a subject to acquire or develop a condition, disease or disorder or the symptoms or complications caused by or associated with the condition, disease or disorder, or to be susceptible to acquiring or developing a condition, disease or disorder or the symptoms or complications cause by or associated with the condition, disease or disorder. In addition, “sensitize” or “sensitizing” may refer to increasing the susceptibility of a subject to acquiring or developing a condition, disease or disorder or the symptoms or complications cause by or associated with the condition, disease or disorder. For example, sensitizing a subject to severe dengue disease upon a secondary or subsequent Dengue virus infection may refer to causing the subject to acquire or develop severe dengue disease or the symptoms or complications caused by or associated with severe dengue disease upon subsequent Dengue virus infection. Sensitizing a subject to severe dengue disease may also refer to causing the subject to be susceptible to acquiring or developing severe dengue disease or one or more other symptoms or complications caused by or associated with severe dengue disease upon a secondary or subsequent Dengue virus infection. In addition, sensitizing a subject to severe dengue disease may also refer to increasing the susceptibility of the subject to acquiring or developing severe dengue disease, one or more other symptoms or complications of severe dengue disease, or more severe symptoms or complications of severe dengue disease, caused by or associated with severe dengue.
A “severe dengue disease” refers to conditions, disease and disorders caused by or associated with Dengue virus infection, including but not limited to dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and any symptoms or complications cause by or associated with DHF and DSS including but not limited to increased vascular permeability, thrombocytopenia, hemorrhagic manifestions and death. In certain embodiments, the development of severe dengue disease may be mediated by antibody dependant enhancement (ADE).
As used herein, the term antibody (Ab) dependent enhancement of infection (ADE) refers to a phenomenon in which a subject who has antibodies against Dengue virus, due to a previous Dengue virus infection or exposure to Dengue virus or antigen (e.g., vaccination, immunization, receipt of maternal anti-Dengue virus antibodies, etc.), suffers from enhanced or a more severe illness after a secondary or subsequent infection with a Dengue virus, or after a Dengue virus vaccination or immunization. Typically, the more severe symptoms include one or more of hemorrhagic fever/Dengue shock syndrome, increased viral load, increased vascular permeability, increased hemorrhagic manifestations, thrombocytopenia, and shock, compared to the acute self-limited illness typically caused by Dengue virus in subjects who have not been vaccinated, immunized or previously infected with Dengue virus. Although not wishing to be bound by any theory, ADE is believed to be a consequence of the presence of serotype cross-reactive antibodies enhancing viral infection of FcγR+ cells resulting in higher Dengue viral loads and a more severe illness upon subsequent exposure or infection of the subject to a Dengue virus or antigen. Methods and uses of the invention therefore include methods and uses that do not substantially or detectably cause, elicit or stimulate one or more symptoms characteristic of ADE, or more broadly ADE, in a subject.
In addition to ADE, there may be other adverse symptoms that result from, or be enhanced or more severe, when a subject who has antibodies against Dengue virus (e.g., due to a prior infection, exposure, vaccination, immunization, maternal antibodies etc.) becomes infected with Dengue virus, or receives a Dengue virus vaccination or immunization, as compared to a subject that has not been vaccinated, immunized or previously infected with a Dengue virus. Such adverse symptoms that may result from, or may be enhanced or more severe include, for example, fever, headache, rash, liver damage, diarrhea, nausea, vomiting or abdominal pain. It is intended that the methods and uses of the invention therefore also include methods and uses that do not substantially elicit, enhance or worsen one or more such other adverse symptoms that may be elicted, enhanced or be more severe in a subject who has antibodies against a Dengue virus, as compared to a subject that does not have antibodies against a Dengue virus.
A Dengue virus protein of the uses, methods and compositions may be a non-structural or structural Dengue virus protein, subsequence or portion or modification thereof. In certain embodiments, the Dengue virus protein is a non-structural Dengue virus protein, for example, NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5. In particular embodiments the Dengue virus protein is a NS3, NS4B or NS5 protein, subsequence or portion or modification thereof. In other embodiments, the Dengue virus protein is a structural Dengue virus protein, for example, Dengue virus envelope protein, membrane protein or core protein, subsequence or portion or modification thereof.
As disclosed herein, a DENV protein, subsequence, portion or modification thereof elicits a cellular or humoral immune response. In particular embodiments, a DENV protein, subsequence, portion or modification thereof, elicits, stimulates, promotes or induces a CD8+ T cell and/or CD4+ T cell response. Such responses can provide protection against (e.g., prophylaxis) an initial DENV infection, or a secondary or subsequent DENV infection. Such T cell responses can also be effective in treatment (e.g., therapeutic) of an initial DENV infection, or a secondary or subsequent DENV infection. Such T cell responses can occur without detectably or substantially eliciting, inducing or promoting severe dengue disease (e.g., ADE mediated DHF or DSS) in a subject having anti-DENV antibodies, or detectably or substantially sensitizing a subject to developing severe dengue disease (e.g., ADE mediated DHF or DSS) upon a subsequent DENV infection.
A DENV protein, subsequence, or portion thereof may be derived from or based upon any sequence from any DENV strain or serotype, such as wild-type. Exemplary serotypes are DENV1, DENV2, DENV3 and DENV4. Thus, in various embodiments, a DENV protein, subsequence, portion or modification thereof is derived from or based upon a DENV1, DENV2, DENV3 or DENV4 sequence. More particularly, a protein, subsequence, portion or modification thereof van be derived from or is based upon West Pacific 74 strain (DENV1), UNC 1017 strain (DENV1), UNC 2005 strain (DENV2), S16803 strain (DENV2), UNC 3001 strain (DENV3), UNC 3043 (DENV3, strain 059.AP-2, Philippines), UNC 3009 strain (DENV3, D2863, Sri Lanka), UNC3066 (DENV3, strain 1342 from Puerto Rico 1977), CH 53489 strain (DENV3), TVP-360 (DENV4), or UNC 4019 strain (DENV4). A DENV protein, subsequence, or portion thereof may also be a modified or variant form (hereinafter referred to as a “modification”). Such modified forms, such as amino acid deletions, additions and substitutions, can also be used in the invention uses, methods and compositions for eliciting, inducing, promoting, increasing or enhancing a T cell response, protecting, vaccinating or immunizing a subject, or treatment of a subject, as set forth herein.
As used herein, a subsequence of a Dengue virus protein includes or consists of one or more amino acids less than the full length Dengue virus protein. The term “subsequence” means a fragment or part of the full length molecule. A subsequence of a Dengue virus protein has one or more amino acids less than the full length Dengue virus protein (e.g. one or more internal or terminal amino acid deletions from either amino or carboxy-termini). Subsequences therefore can be any length up to the full length native molecule, provided said length is at least one amino acid less than full length native molecule.
Subsequences can vary in size, for example, from a polypeptide as small as an epitope capable of binding an antibody (i.e., about five amino acids) up to a polypeptide that is one amino acid less than the entire length of a reference polypeptide such as a Dengue virus protein
In various embodiments, a dengue virus protein subsequence is characterized as including or consisting of a NS1 sequence with less than 380 amino acids in length identical to NS1, a NS2A sequence with less than 159 amino acids in length identical to NS2A, a NS2B sequence with less than 130 amino acids in length identical to NS2B, a NS3 sequence with less than 618 amino acids in length identical to NS3, a NS4A sequence with less than 127 amino acids in length identical to NS4A, a NS4B sequence with less than 248 amino acids in length identical to NS4B, a NS5 sequence with less than 900 amino acids in length identical to NS5, a dengue virus envelope protein sequence with less than 495 amino acids in length identical to dengue virus envelope protein, a dengue virus membrane protein sequence with less than 166 amino acids in length identical to dengue virus membrane protein, a dengue virus core protein sequence with less than 96 amino acids in length identical to dengue virus core protein.
Non-limiting exemplary subsequences less than full length NS 1 sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, or 300 to 380 amino acids in length. Non-limiting exemplary subsequences less than full length NS2A sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 159 amino acids in length. Non-limiting exemplary subsequences less than full length NS2B sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 130 amino acids in length. Non-limiting exemplary subsequences less than full length NS3 sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 618 amino acids in length. Non-limiting exemplary subsequences less than full length NS4A sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 127 amino acids in length. Non-limiting exemplary subsequences less than full length NS4B sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200 to 248 amino acids in length. Non-limiting exemplary subsequences less than full length NS5 sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900 amino acids in length. Non-limiting exemplary subsequences less than full length dengue virus envelope protein sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 495 amino acids in length. Non-limiting exemplary subsequences less than full length dengue virus membrane protein sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 166 amino acids in length. Non-limiting exemplary subsequences less than full length dengue virus core protein sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 96 acids in length.
As used herein, subsequences may also include or consist of one or more amino acid additions or deletions, wherein the subsequence does not comprise the full length native/wild type Dengue virus protein sequence. Accordingly, total subsequence lengths can be greater than the length of the full length native/wild type Dengue virus protein, for example, where a Dengue virus protein subsequence is fused or forms a chimera with another polypeptide.
In other embodiments, the uses, methods and compositions may comprise an Dengue virus protein or peptide comprising or consisting of a subsequence, or an amino acid modification of Dengue virus structural or non-structural protein sequence, wherein the protein or peptide elicits, stimulates, induces, promotes, increases or enhances and anti-Dengue virus CD8+ T cell response or an anti-Dengue virus CD4+ T cell response, as described herein.
A non-limiting example of a protein, subsequence or portion of a Dengue virus (DV) polypeptide sequence includes or consists of a subsequence or portion of Dengue virus (DV) structural Core, Membrane or Envelope polypeptide sequence. A non-limiting example of a protein, subsequence or portion of a Dengue virus (DV) polypeptide sequence includes or consists of a protein, subsequence or portion of Dengue virus (DV) non-structural (NS) NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5 polypeptide sequence.
A non-limiting Core sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting Membrane (M) sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting Envelope (E) sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting non-structural NS1 sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting non-structural NS2A sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting non-structural NS2B sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting non-structural NS3 sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting non-structural NS4A sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting non-structural NS4B sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
A non-limiting non-structural NS5 sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
Structural proteins E and prM are major targets of anti-DENV antibody response. NS proteins (in particular NS3, NS4B and NS5) are more conserved across the four DENV serotypes than E, and NS proteins are not expressed in DENV virions (unlike E and PrM proteins). Thus, without being limited to any particular theory, it appears that NS3, NS4B, or NS5 will be better at inducing cross-protective (heterologous) CD8+ T cell responses and at avoiding ADE. Thus without being limited to or bound by any particular theory, DENV vaccines expressing NS3, NS4B, or NS5 will likely provide superior CD8+ T cell immunity against DENV infection, or secondary or subsequent infection (reinfection) than Envelope and Membrane proteins.
As disclosed herein, Dengue virus (DV) proteins, subsequences, portions and modifications thereof of the invention include those having all or at least partial sequence identity to one or more exemplary Dengue virus (DV) proteins, subsequences, portions or modifications thereof (e.g., sequences set forth in Tables 1-4). The percent identity of such sequences can be as little as 60%, or can be greater (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, etc.). The percent identity can extend over the entire sequence length or a portion of the sequence. In particular aspects, the length of the sequence sharing the percent identity is 2, 3, 4, 5 or more contiguous amino acids, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. contiguous amino acids. In additional particular aspects, the length of the sequence sharing the percent identity is 20 or more contiguous amino acids, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, etc. contiguous amino acids. In further particular aspects, the length of the sequence sharing the percent identity is 35 or more contiguous amino acids, e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 45, 47, 48, 49, 50, etc., contiguous amino acids. In yet further particular aspects, the length of the sequence sharing the percent identity is 50 or more contiguous amino acids, e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110, etc. contiguous amino acids.
The term “identity” and grammatical variations thereof, mean that two or more referenced entities are the same. Thus, where two Dengue virus (DV) proteins, subsequences, portions and modifications thereof are identical, they have the same amino acid sequence. The identity can be over a defined area (region or domain) of the sequence. “Areas, regions or domains” of homology or identity mean that a portion of two or more referenced entities share homology or are the same.
The extent of identity between two sequences can be ascertained using a computer program and mathematical algorithm known in the art. Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region or area. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has exemplary search parameters as follows: Mismatch −2; gap open 5; gap extension 2. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM100, PAM 250, BLOSUM 62 or BLOSUM 50. FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132:185 (2000); and Smith et al., J. Mol. Biol. 147:195 (1981)). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).
In accordance with the invention, modified and variant forms of Dengue virus (DV) proteins, subsequences and portions there are provided. Such forms, referred to as “modifications” or “variants” and grammatical variations thereof, are a Dengue virus (DV) protein, subsequence or portion thereof that deviates from a reference sequence. For example, certain sequences set forth in Tables 1-4 are considered a modification or variant of Dengue virus (DV) protein, subsequence or portion thereof. Such modifications may have greater or less activity or function than a reference Dengue virus (DV) protein, subsequence or portion thereof, such as ability to elicit, stimulate, induce, promote, increase, enhance or activate a CD4+ or a CD8+ T cell response. Thus, Dengue virus (DV) proteins, subsequences and portions thereof include sequences having substantially the same, greater or less relative activity or function as a T cell epitope than a reference T cell epitope (e.g., any of the sequences in Tables 1-4), for example, an ability to elicit, stimulate, induce, promote, increase, enhance or activate an anti-DV CD4+ T cell or anti-DV CD8+ T cell response in vitro or in vivo.
Non-limiting examples of modifications include one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues), additions and insertions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues) and deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100) of a reference Dengue virus (DV) protein, subsequence or portion thereof. In particular embodiments, a modified or variant sequence retains at least part of a function or an activity of unmodified sequence, and can have less than, approximately the same, or greater, but at least a part of, a function or activity of a reference sequence, for example, the ability to elicit, stimulate, induce, promote, increase, enhance or activate an anti-DV CD4+ T cell or anti-DV CD8+ T cell response in vitro or in vivo. Such CD4+ T cell and CD8+ T cell responses elicited include, for example, among others, induced, increased, enhanced, stimulate or activate expression or production of a cytokine (e.g., IFN-gamma, TNF, IL-2 or CD40L), release of a cytotoxin (perforin or granulysin), or apoptosis of a target (e.g., DV infected) cell.
Specific non-limiting examples of substitutions include conservative and non-conservative amino acid substitutions. A “conservative substitution” is the replacement of one amino acid by a biologically, chemically or structurally similar residue. Biologically similar means that the substitution does not destroy a biological activity. Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size. Chemical similarity means that the residues have the same charge, or are both hydrophilic or hydrophobic. Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, and the like.
An addition can be the covalent or non-covalent attachment of any type of molecule to the sequence. Specific examples of additions include glycosylation, acetylation, phosphorylation, amidation, formylation, ubiquitination, and derivatization by protecting/blocking groups and any of numerous chemical modifications. Additional specific non-limiting examples of an addition are one or more additional amino acid residues. Accordingly, DV sequences including DENV proteins, T cell epitopes, subsequences, portions, and modifications thereof can be a part of or contained within a larger molecule, such as another protein or peptide sequence, such as a fusion or chimera with a different DV sequence, or a non-DV protein or subsequence or portion or modification thereof. In particular embodiments, an addition is a fusion (chimeric) sequence, an amino acid sequence having one or more molecules not normally present in a reference native (wild type) sequence covalently attached to the sequence.
The term “chimeric” and grammatical variations thereof, when used in reference to a sequence, means that the sequence contains one or more portions that are derived from, obtained or isolated from, or based upon other physical or chemical entities. For example, a chimera of two or more different proteins may have one part a Dengue virus (DV) peptide, subsequence, portion or modification, and a second part of the chimera may be from a different Dengue virus (DV) protein sequence, or a non-Dengue virus (DV) sequence.
Another particular example of a modified sequence having an amino acid addition is one in which a second heterologous sequence, i.e., heterologous functional domain is attached (covalent or non-covalent binding) that confers a distinct or complementary function. Heterologous functional domains are not restricted to amino acid residues. Thus, a heterologous functional domain can consist of any of a variety of different types of small or large functional moieties. Such moieties include nucleic acid, peptide, carbohydrate, lipid or small organic compounds, such as a drug (e.g., an antiviral), a metal (gold, silver), and radioisotope. For example, a tag such as T7 or polyhistidine can be attached in order to facilitate purification or detection of a T cell epitope. Thus, in other embodiments, the invention provides Dengue virus (DV) proteins, subsequences, portions and modifications thereof and a heterologous domain, wherein the heterologous functional domain confers a distinct function, on the Dengue virus (DV) proteins, subsequences, portions and modifications thereof. Such constructs containing Dengue virus (DV) proteins, subsequences, portions and modifications thereof and a heterologous domain are also referred to as chimeras.
Linkers, such as amino acid or peptidomimetic sequences may be inserted between the sequence and the addition (e.g., heterologous functional domain) so that the two entities maintain, at least in part, a distinct function or activity. Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character, which could promote or interact with either domain. Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. The length of the linker sequence may vary without significantly affecting a function or activity of the fusion protein (see, e.g., U.S. Pat. No. 6,087,329). Linkers further include chemical moieties and conjugating agents, such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST).
Further non-limiting examples of additions are detectable labels. Thus, in another embodiment, the invention provides Dengue virus (DV) proteins, subsequences and portions thereof that are detectably labeled. Specific examples of detectable labels include fluorophores, chromophores, radioactive isotopes (e.g., S35, P32, I125), electron-dense reagents, enzymes, ligands and receptors. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert a substrate such as 3,3-′,5,5-′-tetramethylbenzidine (TMB) to a blue pigment, which can be quantified.
Another non-limiting example of an addition is an insertion of an amino acid within any Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., any DV sequence set forth herein, such as in Tables 1-4). In particular embodiments, an insertion is of one or more amino acid residues inserted into a Dengue virus (DV) protein, subsequence portion or modification thereof, such as any sequence set forth herein, such as in Tables 1-4.
Modified and variant Dengue virus (DV) proteins, subsequences and portions thereof also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms. Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond. Dengue virus (DV) proteins, subsequences and portions thereof may be modified in vitro or in vivo, e.g., post-translationally modified to include, for example, sugar residues, phosphate groups, ubiquitin, fatty acids, lipids, etc.
Specific non-limiting examples of Dengue virus proteinsubsequences or portions include an amino acid sequence comprising at least one amino acid deletion from full length Dengue virus (DV) protein sequence. In particular embodiments, a protein subsequence or portion is from about 5 to 300 amino acids in length, provided that said subsequence or portion is at least one amino acid less in length than the full-length Dengue virus (DV) structural sequence or the non-structural (NS) sequence. In additional particular embodiments, a protein subsequence or portion is from about 2 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 300 amino acids in length, provided that said subsequence or portion is at least one amino acid less in length than the full-length Dengue virus (DV) structural protein sequence or non-structural (NS) protein sequence.
Dengue virus (DV) proteins, subsequences and portions thereof including modified forms can be produced by any of a variety of standard protein purification or recombinant expression techniques. For example, a Dengue virus (DV) protein, subsequence, portion or modification thereof can be produced by standard peptide synthesis techniques, such as solid-phase synthesis. A portion of the protein may contain an amino acid sequence such as a T7 tag or polyhistidine sequence to facilitate purification of expressed or synthesized protein. The protein may be expressed in a cell and purified. The protein may be expressed as a part of a larger protein (e.g., a fusion or chimera) by recombinant methods.
Dengue virus (DV) proteins, subsequences and portions thereof including modified forms can be made using recombinant DNA technology via cell expression or in vitro translation. Polypeptide sequences including modified forms can also be produced by chemical synthesis using methods known in the art, for example, an automated peptide synthesis apparatus (see, e.g., Applied Biosystems, Foster City, Calif.).
The invention provides isolated and/or purified Dengue virus (DV) proteins, including or consisting of a protein, subsequence, portion or modification of a structural core (C), membrane (M) or envelope (E) polypeptide sequence, or a non-structural (NS) NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5 polypeptide sequence. In particular embodiments, an isolated and/or purified protein, subsequence, portion or modification of the Dengue virus (DV) polypeptide sequence includes a T cell epitope, e.g., as set forth in Tables 1-4.
The term “isolated,” when used as a modifier of a composition (e.g., Dengue virus (DV) proteins, subsequences, portions and modifications thereof, nucleic acids encoding same, etc.), means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane. The term “isolated” does not exclude alternative physical forms of the composition, such as fusions/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.
An “isolated” composition (e.g., Dengue virus (DV) protein, subsequence, portion or modification thereof) can also be “substantially pure” or “purified” when free of most or all of the materials with which it typically associates with in nature. Thus, an isolated Dengue virus (DV) protein, subsequence, portion or modification thereof, that also is substantially pure or purified does not include polypeptides or polynucleotides present among millions of other sequences, such as peptides of an peptide library or nucleic acids in a genomic or cDNA library, for example.
A “substantially pure” or “purified” composition can be combined with one or more other molecules. Thus, “substantially pure” or “purified” does not exclude combinations of compositions, such as combinations of Dengue virus (DV) proteins, subsequences, portions and modifications thereof (e.g., multiple, T cell epitopes), and other antigens, agents, drugs or therapies.
The invention also provides nucleic acids encoding Dengue virus (DV) proteins, subsequences, portions and modifications thereof. Such nucleic acid sequences encode a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to a Dengue virus (DV) protein, subsequence or portion thereof. In an additional embodiment, a nucleic acid encodes a sequence having a modification, such as one or more amino acid additions (insertions), deletions or substitutions of a Dengue virus (DV) protein, subsequence or portion thereof, such as any sequence set forth in Tables 1-4.
The terms “nucleic acid,” “polynucleotide” and “polynucleoside” and the like refer to at least two or more ribo- or deoxy-ribonucleic acid base pairs (nucleotides/nucleosides) that are linked through a phosphoester bond or equivalent. Nucleic acids include polynucleotides and polynucleosides. Nucleic acids include single, double or triplex, circular or linear, molecules. Exemplary nucleic acids include but are not limited to: RNA, DNA, cDNA, genomic nucleic acid, naturally occurring and non naturally occurring nucleic acid, e.g., synthetic nucleic acid.
Nucleic acids can be of various lengths. Nucleic acid lengths typically range from about 20 bases to 20 Kilobases (Kb), or any numerical value or range within or encompassing such lengths, 10 bases to 10Kb, 1 to 5 Kb or less, 1000 to about 500 bases or less in length. Nucleic acids can also be shorter, for example, 100 to about 500 bases, or from about 12 to 25, 25 to 50, 50 to 100, 100 to 250, or about 250 to 500 bases in length, or any numerical value or range or value within or encompassing such lengths. In particular aspects, a nucleic acid sequence has a length from about 10-20, 20-30, 30-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-1000, 1000-2000 bases, or any numerical value or range within or encompassing such lengths. Shorter nucleic acids are commonly referred to as “oligonucleotides” or “probes” of single- or double-stranded DNA. However, there is no upper limit to the length of such oligonucleotides.
Nucleic acid sequences further include nucleotide and nucleoside substitutions, additions and deletions, as well as derivatized forms and fusion/chimeric sequences (e.g., encoding recombinant polypeptide). For example, due to the degeneracy of the genetic code, nucleic acids include sequences and subsequences degenerate with respect to nucleic acids that encode Dengue virus (DV) proteins, subsequences and portions thereof, as well as variants and modifications thereof (e.g., substitutions, additions, insertions and deletions).
Nucleic acids can be produced using various standard cloning and chemical synthesis techniques. Techniques include, but are not limited to nucleic acid amplification, e.g., polymerase chain reaction (PCR), with genomic DNA or cDNA targets using primers (e.g., a degenerate primer mixture) capable of annealing to the encoding sequence. Nucleic acids can also be produced by chemical synthesis (e.g., solid phase phosphoramidite synthesis) or transcription from a gene. The sequences produced can then be translated in vitro, or cloned into a plasmid and propagated and then expressed in a cell (e.g., a host cell such as eukaryote or mammalian cell, yeast or bacteria, in an animal or in a plant).
Nucleic acid may be inserted into a nucleic acid construct in which expression of the nucleic acid is influenced or regulated by an “expression control element.” An “expression control element” refers to a nucleic acid sequence element that regulates or influences expression of a nucleic acid sequence to which it is operatively linked. Expression control elements include, as appropriate, promoters, enhancers, transcription terminators, gene silencers, a start codon (e.g., ATG) in front of a protein-encoding gene, etc.
An expression control element operatively linked to a nucleic acid sequence controls transcription and, as appropriate, translation of the nucleic acid sequence. Expression control elements include elements that activate transcription constitutively, that are inducible (i.e., require an external signal for activation), or derepressible (i.e., require a signal to turn transcription off; when the signal is no longer present, transcription is activated or “derepressed”), or specific for cell-types or tissues (i.e., tissue-specific control elements).
Nucleic acid can also be inserted into a plasmid for propagation into a host cell and for subsequent genetic manipulation. A plasmid is a nucleic acid that can be propagated in a host cell, plasmids may optionally contain expression control elements in order to drive expression of the nucleic acid encoding Dengue virus (DV) proteins, subsequences, portions and modifications thereof in the host cell. A vector is used herein synonymously with a plasmid and may also include an expression control element for expression in a host cell (e.g., expression vector). Plasmids and vectors generally contain at least an origin of replication for propagation in a cell and a promoter. Plasmids and vectors are therefore useful for genetic manipulation and expression of Dengue virus (DV) proteins, subsequences and portions thereof. Accordingly, vectors that include nucleic acids encoding or complementary to Dengue virus (DV) proteins, subsequences, portions and modifications thereof, are provided.
In accordance with the invention, there are provided particles (e.g., viral particles) and transformed host cells that express and/or are transformed with a nucleic acid that encodes and/or express Dengue virus (DV) proteins, subsequences, portions and modifications thereof. Particles and transformed host cells include but are not limited to virions, and prokaryotic and eukaryotic cells such as bacteria, fungi (yeast), plant, insect, and animal (e.g., mammalian, including primate and human, CHO cells and hybridomas) cells. For example, bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors; yeast transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus), or transformed animal cell systems engineered for stable expression. The cells may be a primary cell isolate, cell culture (e.g., passaged, established or immortalized cell line), or part of a plurality of cells, or a tissue or organ ex vivo or in a subject (in vivo).
The term “transformed” or “transfected” when used in reference to a cell (e.g., a host cell) or organism, means a genetic change in a cell following incorporation of an exogenous molecule, for example, a protein or nucleic acid (e.g., a transgene) into the cell. Thus, a “transfected” or “transformed” cell is a cell into which, or a progeny thereof in which an exogenous molecule has been introduced by the hand of man, for example, by recombinant DNA techniques.
The nucleic acid or protein can be stably or transiently transfected or transformed (expressed) in the host cell and progeny thereof. The cell(s) can be propagated and the introduced protein expressed, or nucleic acid transcribed. A progeny of a transfected or transformed cell may not be identical to the parent cell, since there may be mutations that occur during replication.
Expression of Dengue virus (DV) proteins, subsequences, portions and modifications thereof, and nucleic acid in particles or introduction into target cells (e.g., host cells) can also be carried out by methods known in the art. Non-limiting examples include osmotic shock (e.g., calcium phosphate), electroporation, microinjection, cell fusion, etc. Introduction of nucleic acid and polypeptide in vitro, ex vivo and in vivo can also be accomplished using other techniques. For example, a polymeric substance, such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers. A nucleic acid can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules, or poly (methylmethacrolate) microcapsules, respectively, or in a colloid system. Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
Liposomes for introducing various compositions into cells are known in the art and include, for example, phosphatidylcholine, phosphatidylserine, lipofectin and DOTAP (e.g., U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282; and GIBCO-BRL, Gaithersburg, Md.). Piperazine based amphilic cationic lipids useful for gene therapy also are known (see, e.g., U.S. Pat. No. 5,861,397). Cationic lipid systems also are known (see, e.g., U.S. Pat. No. 5,459,127). Polymeric substances, microcapsules and colloidal dispersion systems such as liposomes are collectively referred to herein as “vesicles.” Accordingly, viral and non-viral vector means delivery into cells are included.
Dengue virus proteins, subsequences, portions and modifications thereof can be employed in various methods and uses. Such methods and uses include, for example, use, contact or administration of one or more DENV proteins, subsequences or modifications thereof, such as the proteins and subsequences set forth herein (e.g., Tables 1-4), in vitro and in vivo.
In accordance with the invention, there are provided methods for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection, the method comprising administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to elicit an anti-Dengue virus T cell response in the subject.
In another aspect, there is provided a method for providing a subject with protection against a Dengue virus infection or pathology, or one or more physiological disorders, illness, diseases or symptoms caused by or associated with Dengue virus infection or pathology without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection, the method comprising administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to protect the subject against Dengue virus infection.
In yet another aspect of the invention, there is provided a method of vaccinating a subject against a Dengue virus infection without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection, the method comprising administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to vaccinate the subject against the Dengue virus infection.
In a further aspect of the invention, there is provided a method of treating a subject for a Dengue virus infection without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection, the method comprising administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to treat the subject for the Dengue virus infection.
As used herein, the terms “protect” and grammatical variations thereof, when used in reference to a Dengue virus infection or pathology, means preventing a DENV infection, or reducing or decreasing susceptibility to a DENV infection, or preventing or reducing one or more symptoms or pathologies caused by or associated with DENV infection or pathology, such as ADE. A subject may be protected from one or more DENV serotypes, e.g. any or all of DENV 1, 2, 3 or 4, or any variant serotype. A protected subject may also have been previously exposed to or infected with a DENV, and have developed antibodies against DENV. Protection in this context would therefore include, but not be limited to, protection from a secondary or subsequent DENV infection.
In accordance with the invention, uses and methods of stimulating, inducing, promoting, increasing, or enhancing an immune response against Dengue virus (DV) in a subject are provided. In one embodiment, a method includes administering to a subject an amount of a Dengue virus (DV) protein, subsequence or portion or modification thereof, such as a T cell epitope, sufficient to stimulate, induce, promote, increase, or enhance an immune response against Dengue virus (DV) in the subject. Such immune response methods can in turn be used to provide a subject with protection against a Dengue virus (DV) infection or pathology, or one or more physiological conditions, disorders, illness, diseases or symptoms caused by or associated with DV infection or pathology.
In accordance with the invention, treatment uses and methods are provided that include therapeutic (following Dengue virus (DV) infection) and prophylactic (prior to Dengue virus (DV) exposure, infection or pathology) uses and methods. For example, therapeutic and prophylactic uses and methods of treating a subject for a Dengue virus (DV) infection include but are not limited to treatment of a subject having or at risk of having a Dengue virus (DV) infection or pathology, treating a subject with a Dengue virus (DV) infection, and methods of protecting a subject from a Dengue virus (DV) infection (e.g., provide the subject with protection against Dengue virus (DV) infection), to decrease or reduce the probability of a Dengue virus (DV) infection in a subject, to decrease or reduce susceptibility of a subject to a Dengue virus (DV) infection, to inhibit or prevent a Dengue virus (DV) infection in a subject, and to decrease, reduce, inhibit or suppress transmission of the Dengue virus (DV) from a host (e.g., a mosquito) to a subject.
Such methods include, for example, administering Dengue virus (DV) protein, subsequence, portion or modification thereof to therapeutically or prophylactically treat (vaccinate or immunize) a subject having or at risk of having a Dengue virus (DV) infection or pathology. Accordingly, uses and methods can treat a Dengue virus (DV) infection or pathology, or provide a subject with protection from infection (e.g., prophylactic protection).
In one embodiment, a method includes administering to a subject an amount of Dengue virus (DV) protein, subsequence, portion or modification thereof sufficient to treat the subject for the Dengue virus (DV) infection or pathology. In another embodiment, a method includes administering to a subject an amount of a Dengue virus (DV) protein, subsequence, portion or modification sufficient to provide the subject with protection against the Dengue virus (DV) infection or pathology, or one or more physiological conditions, disorders, illness, diseases or symptoms caused by or associated with the virus infection or pathology. In a further embodiment, a method includes administering a subject an amount of a Dengue virus (DV) protein, subsequence, portion or modification sufficient to treat the subject for the Dengue virus (DV) infection.
Dengue virus (DV) proteins, subsequences, portions and modifications thereof include T cell epitopes. In one embodiment, a method includes administering an amount of Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., a T cell epitope) to a subject in need thereof, sufficient to provide the subject with protection against Dengue virus (DV) infection or pathology. In another embodiment, a method includes administering an amount of a Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., a T cell epitope) to a subject in need thereof sufficient to treat, vaccinate or immunize the subject against the Dengue virus (DV) infection or pathology.
In accordance with the invention, uses and methods of inducing, increasing, promoting or stimulating anti-Dengue virus (DV) activity of CD8+ T cells or CD4+ T cells in a subject are provided. In one embodiment, a method includes administering to a subject an amount of a Dengue virus (DV) protein, subsequence or portion, or modification thereof, such as a T cell epitope, sufficient to induce, increase, promote or stimulate anti-Dengue virus (DV) activity of CD8+ T cells or CD4+ T cells in the subject.
In methods of the invention, any appropriate Dengue virus (DV) protein, subsequence, portion or modification thereof can be used or administered. Non-limiting examples include Dengue virus (DV) protein, subsequence, portion or modification thereof of a DENV1, DENV2, DENV3 or DENV4 serotype protein, subsequence or portion or modification thereof, such as a T cell epitope. Additional non-limiting examples include a Dengue virus structural protein (e.g., C, M or E) or non-structural (NS) protein (e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B or NS5), or a subsequence or portion or modification thereof, such as a T cell epitope, in or of such structural and non-structural (NS) proteins. Particular non-limiting examples include a DENV protein, or a protein subsequence, such as a sequence set forth in Tables 1-4, or a subsequence or a modification thereof.
In particular uses and methods embodiments, one or more disorders, diseases, physiological conditions, pathologies and symptoms associated with or caused by a Dengue virus (DV) infection or pathology will respond to treatment. In particular methods embodiments, treatment uses and methods reduce, decrease, suppress, limit, control or inhibit Dengue virus (DV) numbers or titer; reduce, decrease, suppress, limit, control or inhibit pathogen proliferation or replication; reduce, decrease, suppress, limit, control or inhibit the amount of a pathogen protein; or reduce, decrease, suppress, limit, control or inhibit the amount of a Dengue virus (DV) nucleic acid. In additional particular uses and methods embodiments, treatment uses and methods include an amount of a Dengue virus (DV) protein, subsequence or portion or modification thereof sufficient to increase, induce, enhance, augment, promote or stimulate an immune response against a Dengue virus (DV); increase, induce, enhance, augment, promote or stimulate Dengue virus (DV) clearance or removal; or decrease, reduce, inhibit, suppress, prevent, control, or limit transmission of Dengue virus (DV) to a subject (e.g., transmission from a host, such as a mosquito, to a subject). In further particular uses and methods embodiments, treatment uses and methods include an amount of Dengue virus (DV) protein, subsequence or portion or modification thereof sufficient to protect a subject from a Dengue virus (DV) infection or pathology, or reduce, decrease, limit, control or inhibit susceptibility to Dengue virus (DV) infection or pathology.
Uses and methods of the invention include treatment uses and methods, which result in any therapeutic or beneficial effect. In various methods embodiments, Dengue virus (DV) infection, proliferation or pathogenesis is reduced, decreased, inhibited, limited, delayed or prevented, or a use or method decreases, reduces, inhibits, suppresses, prevents, controls or limits one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology (e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite). In additional various particular embodiments, treatment uses and methods include reducing, decreasing, inhibiting, delaying or preventing onset, progression, frequency, duration, severity, probability or susceptibility of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology (e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite). In further various particular embodiments, treatment uses and methods include improving, accelerating, facilitating, enhancing, augmenting, or hastening recovery of a subject from a Dengue virus (DV) infection or pathogenesis, or one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology (e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite). In yet additional various embodiments, treatment uses and methods include stabilizing infection, proliferation, replication, pathogenesis, or an adverse symptom, disorder, illness, disease or complication caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology, or decreasing, reducing, inhibiting, suppressing, limiting or controlling transmission of Dengue virus (DV) from a host (e.g., mosquito) to an uninfected subject.
A therapeutic or beneficial effect of treatment is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject. A therapeutic or beneficial effect can but need not be complete ablation of all or any particular adverse symptom, disorder, illness, disease or complication caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology (e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite). Thus, a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in an adverse symptom, disorder, illness, disease or complication caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection, Dengue virus (DV) numbers, titers, proliferation or replication, Dengue virus (DV) protein or nucleic acid, or Dengue virus (DV) pathology, over a short or long duration (hours, days, weeks, months, etc.).
A therapeutic or beneficial effect also includes reducing or eliminating the need, dosage frequency or amount of a second active such as another drug or other agent (e.g., anti-viral) used for treating a subject having or at risk of having a Dengue virus (DV) infection or pathology. For example, reducing an amount of an adjunct therapy, for example, a reduction or decrease of a treatment for a Dengue virus (DV) infection or pathology, or a vaccination or immunization protocol is considered a beneficial effect. In addition, reducing or decreasing an amount of a Dengue virus (DV) antigen used for vaccination or immunization of a subject to provide protection to the subject is considered a beneficial effect.
Adverse symptoms and complications associated with Dengue virus (DV) infection and pathology include, for example, e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite, etc. Thus, the aforementioned symptoms and complications are treatable in accordance with the invention. Other symptoms of Dengue virus (DV) infection and pathology include ADE, which occurs upon a secondary or subsequent DENV infection of a subject, which had been previously infected with or exposed to DENV. ADE, as set forth herein or known to one of skill in the art, can be minimized or avoided (i.e., a subject would not be sensitized to ADE), or ADE would not be substantially elicited, induced, stimulated or promoted in a subject, in accordance with the invention uses and methods. Additional symptoms of Dengue virus (DV) infection or pathogenesis are known to one of skill in the art and treatment thereof in accordance with the invention is provided.
Uses, methods and compositions of the invention also include increasing, stimulating, promoting, enhancing, inducing or augmenting an anti-DENV CD4+ and/or CD8+ T cell responses in a subject, such as a subject with or at risk of a Dengue virus infection or pathology. In one embodiment, a use or method includes administering to a subject an amount of Dengue virus (DV) protein, subsequence, portion or modification thereof sufficient to increase, stimulate, promote, enhance, augment or induce anti-DENV CD4+ or CD8+T cell response in the subject. In another embodiment, a method includes administering to a subject an amount of Dengue virus (DV) protein, subsequence, portion or modification thereof, and administering a Dengue virus (DV) antigen, live or attenuated Dengue virus (DV), or a nucleic acid encoding all or a portion (e.g., a T cell epitope) of any protein or proteinaceous Dengue virus (DV) antigen sufficient to increase, stimulate, promote, enhance, augment or induce anti-Dengue virus (DV) CD4+ T cell or CD8+ T cell response in the subject.
Uses and methods of the invention additionally include, among other things, increasing production of a Th1 cytokine (e.g., IFN-gamma, TNF-alpha, IL-1alpha, IL-2, IL-6, IL-8, etc.) or other signaling molecule (e.g., CD40L) in vitro or in vivo. In one embodiment, a method includes administering to a subject in need thereof an amount of Dengue virus (DV) protein, subsequence or portion or modification thereof sufficient to increase production of a Th1 cytokine in the subject (e.g., IFN-gamma, TNF-alpha, IL-lalpha, IL-2, IL-6, IL-8, etc.) or other signaling molecule (e.g., CD40L).
Uses, methods and compositions of the invention include administration of Dengue virus (DV) protein, subsequence, portion or modification thereof to a subject prior to contact, exposure or infection by a Dengue virus, administration prior to, substantially contemporaneously with or after a subject has been contacted by, exposed to or infected with a Dengue virus (DV), and administration prior to, substantially contemporaneously with or after Dengue virus (DV) pathology or development of one or more adverse symptoms. Methods, compositions and uses of the invention also include administration of Dengue virus (DV) protein, subsequence, portion or modification thereof to a subject prior to, substantially contemporaneously with or following an adverse symptom, disorder, illness or disease caused by or associated with a Dengue virus (DV) infection, or pathology. A subject infected with a Dengue virus (DV) may have an infection over a period of 1-5, 5-10, 10-20, 20-30, 30-50, 50-100 hours, days, months, or years.
Invention compositions (e.g., Dengue virus (DV) protein, subsequence or portion or modification thereof, including T cell epitopes) and uses and methods can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include multiple DENV proteins, subsequences, portions or modifications thereof, such as T cell epitopes as set for the herein, second actives, such as anti-Dengue virus (DV) compounds, agents and drugs, as well as agents that assist, promote, stimulate or enhance efficacy. Such anti-Dengue virus (DV) drugs, agents, treatments and therapies can be administered or performed prior to, substantially contemporaneously with or following any other use or method of the invention, for example, a therapeutic use or method of treating a subject for a Dengue virus (DV) infection or pathology, or a use or method of prophylactic treatment of a subject for a Dengue virus (DV) infection.
Dengue virus (DV) proteins, subsequences, portions and modifications thereof can be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) administering a second active, to a subject. The invention therefore provides combinations in which a use or method of the invention is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, such as an anti-viral (e.g., Dengue virus (DV)) or immune stimulating, enhancing or augmenting protocol, or pathogen vaccination or immunization (e.g., prophylaxis) set forth herein or known in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of one or more Dengue virus (DV) proteins, subsequences, portions or modifications thereof, or a nucleic acid encoding all or a portion (e.g., a T cell epitope) of a Dengue virus (DV) protein, subsequence, portion or modification thereof, to a subject. Specific non-limiting examples of combination embodiments therefore include the foregoing or other compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition.
An exemplary combination is a Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., a CD4+ or CD8+ T cell epitope) and a different Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., a different T cell epitope) such as a DENV protein or T cell epitope, antigen (e.g., Dengue virus (DV) extract), or live or attenuated Dengue virus (DV) (e.g., inactivated Dengue virus (DV)). Another exemplary combination is a Dengue virus (DV) protein, subsequence, portion or modification thereof and a T-cell stimulatory molecule, including for example an OX40 or CD27 agonist.
Such Dengue virus (DV) proteins, antigens and T cell epitopes set forth herein or known to one skilled in the art include Dengue virus (DV) proteins and antigens that increase, stimulate, enhance, promote, augment or induce a proinflammatory or adaptive immune response, numbers or activation of an immune cell (e.g., T cell, natural killer T (NKT) cell, dendritic cell (DC), B cell, macrophage, neutrophil, eosinophil, mast cell, CD4+ or a CD8+ cell, B220+ cell, CD14+, CD11b+ or CD11c+ cells), an anti-Dengue virus (DV) CD4+ or CD8+ T cell response, production of a Th1 cytokine, a T cell mediated immune response, such as activation of CD8+ T cells, or induction of CD8+ memory T cells, etc.
Combination methods and use embodiments include, for example, second actives such as anti-pathogen drugs, such as protease inhibitors, reverse transcriptase inhibitors, virus fusion inhibitors and virus entry inhibitors, antibodies to pathogen proteins, live or attenuated pathogen, or a nucleic acid encoding all or a portion (e.g., an epitope) of any protein or proteinaceous pathogen antigen, immune stimulating agents, etc., and include contact with, administration in vitro or in vivo, with another compound, agent, treatment or therapeutic regimen appropriate for pathogen infection, vaccination or immunization
Uses and methods of the invention also include, among other things, uses and methods that result in a reduced need or use of another compound, agent, drug, therapeutic regimen, treatment protocol, process, or remedy. For example, for a treatment of Dengue virus (DV) infection or pathology, or vaccination or immunization, a use or method of the invention has a therapeutic benefit if in a given subject a less frequent or reduced dose or elimination of an anti-Dengue virus (DV) treatment results. Thus, in accordance with the invention, uses and methods of reducing need or use of a treatment or therapy for a Dengue virus (DV) infection or pathology, or vaccination or immunization, are provided.
In invention uses and methods in which there is a desired outcome, such as a therapeutic or prophylactic method that provides a benefit from treatment, vaccination or immunization, a Dengue virus (DV) protein, subsequence, portion or modification thereof can be administered in a sufficient or effective amount.
As used herein, a “sufficient amount” or “effective amount” or an “amount sufficient” or an “amount effective” refers to an amount that provides, in single (e.g., primary) or multiple (e.g., booster) doses, alone or in combination with one or more other compounds, treatments, therapeutic regimens or agents (e.g., a drug), a long term or a short term detectable or measurable improvement in a given subject or any objective or subjective benefit to a given subject of any degree or for any time period or duration (e.g., for minutes, hours, days, months, years, or cured).
An amount sufficient or an amount effective can but need not be provided in a single administration and can but need not be achieved by Dengue virus (DV) protein, subsequence, portion or modification thereof alone, optionally in a combination composition or method that includes a second active. In addition, an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second or additional administration or dosage, since additional doses, amounts or duration above and beyond such doses, or additional antigens, compounds, drugs, agents, treatment or therapeutic regimens may be included in order to provide a given subject with a detectable or measurable improvement or benefit to the subject. For example, to increase, enhance, improve or optimize immunization and/or vaccination, after an initial or primary administration of one or more Dengue virus (DV) proteins, subsequences, portions or modifications thereof to a subject, the subject can be administered one or more additional “boosters” of one or more Dengue virus (DV) proteins, subsequences, portions or modifications thereof. Such subsequent “booster” administrations can be of the same or a different formulation, dose or concentration, route, etc.
An amount sufficient or an amount effective need not be therapeutically or prophylactically effective in each and every subject treated, nor a majority of subjects treated in a given group or population. An amount sufficient or an amount effective means sufficiency or effectiveness in a particular subject, not a group of subjects or the general population. As is typical for such methods, different subjects will exhibit varied responses to a use or method of the invention, such as immunization, vaccination and therapeutic treatments.
The term “subject” refers to a subject at risk of DENV exposure or infection as well as a subject that has been exposed or already infected with DENV. Such subjects, include mammalian animals (mammals), such as a non human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
Subjects include animal disease models, for example, mouse and other animal models of pathogen (e.g., DENV) infection known in the art.
Accordingly, subjects appropriate for treatment include those having or at risk of exposure to Dengue virus infection or pathology, also referred to as subjects in need of treatment. Subjects in need of treatment therefore include subjects that have been exposed to or contacted with Dengue virus (DV), or that have an ongoing infection or have developed one or more adverse symptoms caused by or associated with Dengue virus (DV) infection or pathology, regardless of the type, timing or degree of onset, progression, severity, frequency, duration of the symptoms.
Target subjects and subjects in need of treatment also include those at risk of Dengue virus (DV) exposure, contact, infection or pathology or at risk of having or developing a Dengue virus (DV) infection or pathology. The invention uses, methods and compositions are therefore applicable to treating a subject who is at risk of Dengue virus (DV) exposure, contact, infection or pathology, but has not yet been exposed to or contacted with Dengue virus (DV). Prophylactic uses and methods are therefore included. Target subjects for prophylaxis can be at increased risk (probability or susceptibility) of exposure, contact, infection or pathology, as set forth herein. Such subjects are considered in need of treatment due to being at risk.
Subjects for prophylaxis need not be at increased risk but may be from the general population in which it is desired to vaccinate or immunize a subject against a Dengue virus (DV) infection, for example. Such a subject that is desired to be vaccinated or immunized against a Dengue virus (DV) can be administered Dengue virus (DV) protein, subsequence, portion or modification thereof. In another non-limiting example, a subject that is not specifically at risk of exposure to or contact with a Dengue virus (DV), but nevertheless desires protect against infection or pathology, can be administered a Dengue virus (DV) protein, subsequence, portion or modification thereof. Such subjects are also considered in need of treatment.
At risk subjects appropriate for treatment also include subjects exposed to environments in which subjects are at risk of a Dengue virus (DV) infection due to mosquitos. Subjects appropriate for treatment therefore include human subjects exposed to mosquitos, or travelling to geographical regions or countries in which Dengue virus (DV) is known to infect subjects, for example, an individual who risks exposure due to the presence of DENV in a particular geographical region or country or population, or transmission from mosquitos present in the region or country. At risk subjects appropriate for treatment also include subjects where the risk of Dengue virus (DV) infection or pathology is increased due to changes in infectivity or the type of region of Dengue virus (DV) carrying mosquitos. Such subjects are also considered in need of treatment due to such a risk.
“Prophylaxis” and grammatical variations thereof mean a use or a method in which contact, administration or in vivo delivery to a subject is prior to contact with or exposure to DENV or DENV infection. In certain situations it may not be known that a subject has been contacted with or exposed to Dengue virus (DV), but administration or in vivo delivery to a subject can be performed prior to infection or manifestation of pathology (or an associated adverse symptom, condition, complication, etc. caused by or associated with a Dengue virus (DV)). For example, a subject can be immunized or vaccinated with a Dengue virus (DV) protein, subsequence, portion or modification thereof. In such case, a use or method can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a Dengue virus (DV) infection or pathology, or an adverse symptom, condition or complication associated with or caused by or associated with a Dengue virus (DV) infection or pathology.
“Prophylaxis” can also refer to a use or a method in which contact, administration or in vivo delivery to a subject is prior to a secondary or subsequent exposure or infection. In such a situation, a subject may have had a prior DENV infection, or have been contacted with or exposed to Dengue virus (DV). In such subjects, an acute DENV infection may but not need be resolved. Such a subject typically has developed anti-DENV antibodies due to the prior exposure or infection. Immunization or vaccination, by administration or in vivo delivery to such a subject, can be performed prior to a secondary or subsequent DENV infection or exposure. Such a use or method can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a secondary or subsequent Dengue virus (DV) infection or pathology, or an adverse symptom, condition or complication associated with or caused by or associated with a Dengue virus (DV) infection or pathology, or an adverse symptom or pathology associated with the development of anti-DENV antibodies, such as ADE.
Treatment of an infection can be at any time during the infection. Dengue virus (DV) protein, subsequence or portion or modification thereof can be administered as a combination (e.g., with a second active), or separately concurrently or in sequence (sequentially) in accordance with the uses and methods as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by Dengue virus (DV) infection, pathology, or an adverse symptom, condition or complication associated with or caused by a Dengue virus (DV). Thus, a method can be practiced one or more times (e.g., 1-10, 1-5 or 1-3 times) an hour, day, week, month, or year. The skilled artisan will know when it is appropriate to delay or discontinue administration. A non-limiting dosage schedule is 1-7 times per week, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.
Uses and methods of the invention may be practiced by any mode of administration or delivery, or by any route, systemic, regional and local administration or delivery. Exemplary administration and delivery routes include intravenous (i.v.), intraperitoneal (i.p.), intrarterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, intra-cranial, intra-spinal, rectal, oral (alimentary), mucosal, inhalation, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, or intralymphatic.
Doses can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies set forth herein, for a mouse, which weighs about 30 grams, and the amount of Dengue virus (DV) protein, subsequence, portion or modification thereof administered that is determined to be effective. Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg/kg to about 100 mg/kg, and any numerical value or range or value within such ranges. Greater or lesser amounts (doses) can be administered, for example, 0.01-500 mg/kg, and any numerical value or range or value within such ranges. The dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1-10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50-100 ug/kg,100-500 ug/kg, 500-1,000 ug/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, two, three, four, or more times per hour, day, week, month or annually. A typical range will be from about 0.3 mg/kg to about 50 mg/kg, 0-25 mg/kg, or 1.0-10 mg/kg, or any numerical value or range or value within such ranges.
Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has been previously exposed to, infected with our suffered from Dengue virus (DV), the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, condition, pathology or complication, or vaccination or immunization to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.
Typically, for treatment, Dengue virus (DV) protein, subsequence, portion or modification thereof will be administered as soon as practical, typically within 1-2, 2-4, 4-12, 12-24 or 24-72 hours after a subject is exposed to or contacted with a Dengue virus (DV), or within 1-2, 2-4, 4-12, 12-24 or 24-48 hours after onset or development of one or more adverse symptoms, conditions, pathologies, complications, etc., associated with or caused by a Dengue virus (DV) infection or pathology. For prophylactic treatment in connection with vaccination or immunization, Dengue virus (DV) protein, subsequence, portion or modification thereof can be administered for a duration of 0-4 weeks, e.g., 2-3 weeks, prior to exposure to, contact or infection with Dengue virus (DV), or at least within 1-2, 2-4, 4-12, 12-24, 24-48 or 48-72 hours prior to exposure to, contact or infection with Dengue virus (DV). For an acute infection, Dengue virus (DV) protein, subsequence, portion or modification thereof is administered at any appropriate time.
The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by the status of the subject. For example, whether the subject has a pathogen infection, whether the subject has been exposed to, contacted or infected with pathogen or is merely at risk of pathogen contact, exposure or infection, whether the subject is a candidate for or will be vaccinated or immunized. The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy.
In the uses and methods of the invention, the route, dose, number and frequency of administrations, treatments, immunizations or vaccinations, and timing/intervals between treatment, immunization and vaccination, and viral challenge can be modified. Although rapid induction of immune responses is desired for developing protective emergency vaccines against DENV, in certain embodiments, a desirable DENV vaccine will elicit robust, long-lasting immunity. Thus, in certain embodiments, invention uses, methods and compositions provide long-lasting immunity to DENV. Immunization strategies provided can provide long-lived protection against DENV challenge, depending on the level of vaccine-induced CD8+ T cell response.
The invention also provides an amount of a Dengue virus protein, subsequence or portion, or modification thereof for use in: eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection; providing a subject with protection against a Dengue virus infection or pathology, or one or more physiological disorders, illness, diseases or symptoms caused by or associated with Dengue virus infection or pathology without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection; vaccinating a subject against a Dengue virus infection without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection; and treating a subject for a Dengue virus infection without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection. In certain embodiments, DENV proteins, subsequences, portions and modifications thereof may be pharmaceutical compositions.
As used herein the term “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.
Pharmaceutical compositions can be formulated to be compatible with a particular route of administration. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. Exemplary routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, opthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.
Formulations suitable for parenteral administration comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
To increase an immune response, immunization or vaccination, Dengue virus (DV) proteins, subsequences, portions and modifications thereof can be coupled to another protein such as ovalbumin or keyhole limpet hemocyanin (KLH), thyroglobulin or a toxin such as tetanus or cholera toxin. Dengue virus (DV) proteins, subsequences, portions and modifications thereof can also be mixed with adjuvants.
Adjuvants include, for example: Oil (mineral or organic) emulsion adjuvants such as Freund's complete (CFA) and incomplete adjuvant (IFA) (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241; and U.S. Pat. No. 5,422,109); metal and metallic salts, such as aluminum and aluminum salts, such as aluminum phosphate or aluminum hydroxide, alum (hydrated potassium aluminum sulfate); bacterially derived compounds, such as Monophosphoryl lipid A and derivatives thereof (e.g., 3 De-O-acylated monophosphoryl lipid A, aka 3D-MPL or d3-MPL, to indicate that position 3 of the reducing end glucosamine is de-O-acylated, 3D-MPL consisting of the tri and tetra acyl congeners), and enterobacterial lipopolysaccharides (LPS); plant derived saponins and derivatives thereof, for example Quil A (isolated from the Quilaja Saponaria Molina tree, see, e.g., “Saponin adjuvants”, Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254; U.S. Pat. No. 5,057,540), and fragments of Quil A which retain adjuvant activity without associated toxicity, for example QS7 and QS21 (also known as QA7 and QA21), as described in WO96/33739, for example; surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone; oligonucleotides such as CpG (WO 96/02555, and WO 98/16247), polyriboA and polyriboU; block copolymers; and immunostimulatory cytokines such as GM-CSF and IL-1, and Muramyl tripeptide (MTP). Additional examples of adjuvants are described, for example, in “Vaccine Design—the subunit and adjuvant approach” (Edited by Powell, M. F. and Newman, M. J.; 1995, Pharmaceutical Biotechnology (Plenum Press, New York and London, ISBN 0-306-44867-X) entitled “Compendium of vaccine adjuvants and excipients” by Powell, M. F. and Newman M.
Cosolvents may be added to a Dengue virus (DV) protein, subsequence, portion or modification composition or formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
Supplementary compounds (e.g., preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions may therefore include preservatives, anti-oxidants and antimicrobial agents.
Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation. Suitable preservatives are known in the art and include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate. Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.
An antimicrobial agent or compound directly or indirectly inhibits, reduces, delays, halts, eliminates, arrests, suppresses or prevents contamination by or growth, infectivity, replication, proliferation, reproduction, of a pathogenic or non-pathogenic microbial organism. Classes of antimicrobials include antibacterial, antiviral, antifungal and antiparasitics. Antimicrobials include agents and compounds that kill or destroy (-cidal) or inhibit (-static) contamination by or growth, infectivity, replication, proliferation, reproduction of the microbial organism.
Exemplary antibacterials (antibiotics) include penicillins (e.g., penicillin G, ampicillin, methicillin, oxacillin, and amoxicillin), cephalosporins (e.g., cefadroxil, ceforanid, cefotaxime, and ceftriaxone), tetracyclines (e.g., doxycycline, chlortetracycline, minocycline, and tetracycline), aminoglycosides (e.g., amikacin, gentamycin, kanamycin, neomycin, streptomycin, netilmicin, paromomycin and tobramycin), macrolides (e.g., azithromycin, clarithromycin, and erythromycin), fluoroquinolones (e.g., ciprofloxacin, lomefloxacin, and norfloxacin), and other antibiotics including chloramphenicol, clindamycin, cycloserine, isoniazid, rifampin, vancomycin, aztreonam, clavulanic acid, imipenem, polymyxin, bacitracin, amphotericin and nystatin.
Particular non-limiting classes of anti-virals include reverse transcriptase inhibitors; protease inhibitors; thymidine kinase inhibitors; sugar or glycoprotein synthesis inhibitors; structural protein synthesis inhibitors; nucleoside analogues; and viral maturation inhibitors. Specific non-limiting examples of anti-virals include nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, zidovudine (AZT), stavudine (d4T), larnivudine (3TC), didanosine (DDI), zalcitabine (ddC), abacavir, acyclovir, penciclovir, ribavirin, valacyclovir, ganciclovir, 1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9->2-hydroxy-ethoxy methylguanine, adamantanamine, 5-iodo-2′-deoxyuridine, trifluorothymidine, interferon and adenine arabinoside.
Pharmaceutical formulations and delivery systems appropriate for the compositions and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel ad Soklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).
Dengue virus (DV) proteins, subsequences, portions, and modifications thereof, along with any adjunct agent, compound drug, composition, whether active or inactive, etc., can be packaged in unit dosage form (capsules, tablets, troches, cachets, lozenges) for ease of administration and uniformity of dosage. A “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms also include, for example, ampules and vials, which may include a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein. Individual unit dosage forms can be included in multi-dose kits or containers. Pharmaceutical formulations can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
All applications, publications, patents and other references, GenBank citations and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.
As used herein, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a “Dengue virus (DV) protein, subsequence, portion, or modification thereof,” or a “Dengue virus (DV)” includes a plurality of Dengue virus (DV) proteins, subsequences, portions, and modifications thereof, such as CD4+ and/or CD8+ T cell epitopes, or serotypes of Dengue virus (DV), and reference to an “activity or function” can include reference to one or more activities or functions of a Dengue virus (DV) protein, subsequence, portion, or modification thereof, including function as a T cell epitopes, an ability to elicit, stimulate, induce, promote, increase, enhance or activate a measurable or detectable anti-DV CD4+ T cell response or anti-DV CD8+ T cell response, and so forth.
As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, to illustrate, reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Reference to a range of 1-5 fold therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth. Further, for example, reference to a series of ranges of 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours and 6-12 hours, includes ranges of 2-6 hours, 2, 12 hours, 2-18 hours, 2-24 hours, etc., and 4-27 hours, 4-48 hours, 4-6 hours, etc.
As also used herein a series of range formats are used throughout this document. The use of a series of ranges includes combinations of the upper and lower ranges to provide a range. Accordingly, a series of ranges include ranges which combine the values of the boundaries of different ranges within the series. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, and 150-171, includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, 5-171, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, 10-171, and 20-40, 20-50, 20-75, 20-100, 20-150, 20-171, and so forth.
The invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. For example, in certain embodiments or aspects of the invention, antibodies or other materials and method steps are excluded. In certain embodiments and aspects of the invention, for example, a Dengue virus (DV) protein, subsequence, portion, or modification thereof, is excluded. Thus, even though the invention is generally not expressed herein in terms of what is not included, embodiments and aspects that expressly exclude compositions or method steps are nevertheless disclosed and included in the invention.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims.
This example includes a description of an ADE mouse model that reflects ADE in humans.
Antibody (Ab)-induced dengue disease is a severe condition that affects humans having existing Dengue virus antibodies. A clinically relevant model of antibody (Ab)-induced dengue disease (ADE) in mice is disclosed. The model demonstrates, for the first time, ADE in vivo (Zellweger, et al. Cell Host Microbe 7:128-139 (2010)).
Briefly, AG129 mice were passively administered 15 μg of mouse mAb of subclass IgG2a (clone 2H2; DENV1-4 cross-reactive) before infection with 5×108 genomic equivalents (GE) (≈104 PFU) of the DENV2 strain S221. Mice treated with 2H2 succumbed early to S221 infection (day 4-6) and featured the hallmarks of severe dengue disease in humans (high viral load, elevated hematocrit, cytokine storm, low platelet count, increased vascular permeability, hemorrhagic manifestations, and shock-induced death). In contrast, mice treated with isotype control Ab developed paralysis at later times after infection (day 10-30).
This example includes data demonstrating that vaccination with inactivated Dengue virus mediates ADE.
The demonstration of ADE in the clinically relevant animal model of human ADE allows the evaluation of aspects of protective versus pathogenic effects of dengue vaccination. Three general types of dengue vaccines are currently under development, inactivated, subviral particles or subunit, and live attenuated (Murphy, et al. Ann. Rev. of Immunol. 29:587-619 (2011)). First assessed was whether UV-inactivated DENV2 in alum can mediate protection as an inactivated vaccine candidate. Alum was chosen as the adjuvant because it is used in many human vaccines and is known to promote humoral immunity, which is believed to be required for dengue vaccine-mediated protection.
AG129 mice were injected with 1011 GE (≈2×106 PFU) of UV-inactivated DENV2 strain S221 via a subcutaneous (s.c.) or intraperitoneal (i.p.) route 14 and 5 days before a sublethal intravenous (i.v.) infection with S221 (5 ×108 GE or ≈104 PFU) (schematized in
DENV RNA levels in the liver at day 3 after viral challenge were measured by qRT-PCR analysis. As expected, control mice with enhancing Ab (i.e. the ADE group) contained ≈10 fold-higher viral RNA levels than the baseline/isotype group (
To confirm that antibodies were responsible for the high viral load in the liver of UV-inactivated DENV2 in alum-vaccinated mice, serum from immunized mice was passively transferred i.v. into naïve mice 1 day before viral challenge. Mice administered the immunized mouse serum had elevated levels of DENV RNA in the liver at day 3 post-challenge (
This example includes data demonstrating that Dengue Virus protein can provide protective immunity, without substantially inducing ADE, and even in the presence of enhancing antibodies.
To ascertain the ability of a DENV2 envelope (E) protein to provide protection against Dengue virus, non-propagating Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRP) coding for DENV2 envelope (E) protein (i.e. VRP-DENV2E) were used. UV-inactivated DENV2 plus alum regimen is shown in
To explore the nature of DENV2E-mediated protection of mice, it was determined whether vaccination would provide protective immunity upon ADE challenge. AG129 mice were immunized with VRP-DENV2E on 14 and 5 days before viral challenge (i.e. the same immunization protocol as all studies described thus far), but the immunized mice were administered anti-DENV mAb (15 μg of clone 2H2) just prior to i.v. inoculation with S221. It was found that DENV2E-vaccination reduced viral RNA levels in the liver on day 3 after challenge with virus alone or with virus plus anti-DENV Ab, indicating that DENV2 immunization strategy offers protection even in the presence of enhancing Abs (
In the animal studies described in White, et al., supra the animals do not and are not capable of developing ADE. Thus, in contrast to the model disclosed herein which develops ADE, the animal model in White, et al., supra does not reflect human DENV infection, particularly humans previously infected with or exposed to DENV that have developed anti-DENV antibodies and are therefore at risk of ADE upon subsequent infection or exposure to DENV. Furthermore, the studies in White, et al. are limited to analysis of anti-DENV antibodies that purportedly provide protection, but as disclosed herein antibodies exacerbate Dengue virus illness upon a secondary or subsequent DENV infection or exposure of an individual who has developed such anti-DENV antibodies, resulting in ADE. Moreover, subsequent studies indicated that tetravalent immunization with all four Dengue virus serotypes is required to produce a broad spectrum antibody response, which antibodies were merely shown to be capable of neutralizing Dengue virus in vitro, but not broad spectrum protection against two or more DENV serotypes, from infection and/or symptoms associated with or caused by DENV infection, and do not demonstrate a lack of producing substantial ADE, or eliciting, inducing or promoting ADE, since such studies are in animals that do not develop ADE and therefore are not reflective of DENV infection in humans, particularly those that have developed antibodies to one or more DENV serotypes and are therefore at risk of ADE.
This example includes data demonstrating that cell-mediated immunity contributes to the DENV2E-mediated protection against DENV.
To analyze the mechanisms by which the DENV2E vaccination provides protective immunity, antibody responses induced by the two qualitatively different vaccine candidates (UV-inactivated 5221 plus alum compared to VRP-DENV2E) were first compared. One day before viral challenge, serum samples were collected from the immunized mice that were used for studies in
Although direct comparison of DENV-specific IgG levels between the two groups of immunized mice is not feasible due to the presence of different antigens in UV-inactivated virus (which contains both prM/M and E protein) versus DENV2E (which contains only E), both vaccine candidates induced DENV-specific binding Abs in all immunized mice (
This example includes data demonstrating that CD8+ T cells provide early protective capacity against Dengue virus.
To measure the contribution of T cells in DENV2E vaccine-mediated protection, mice were immunized with VRP-DENV2E as described above, followed by depletion of CD4+ and/or CD8+ T cells before challenge with S221 (
Studies examining heterologous DENV infections were also conducted. Following infection with live DENV3 (UNC3001), CD8+ T cells were depleted in mice by administration of an anti-CD8+ antibody, as discussed herein. The mice were then infected with live DENV 2 (S221). DENV2 viral RNA levels in the liver of mice were determined by qRT-PCR (
This Example includes studies demonstrating that adoptively transferred wild-type T cells protect against DENV in AG129 mice.
Wild-type 129/Sv mice were immunized i.p. with 106 GE of VRP-DENV2E on days −14 and −5, followed by isolation of total T cells (both CD4+ and CD8+) by MACS negative selection on day 0 and i.v. transfer into AG129 mice (
As disclosed herein, the data indicate a rapidly protective, CD8+ T cell-dependent DENV immunization strategy using DENV2E in a clinically relevant model of DENV infection. Although fast induction of immune responses is important for developing protective emergency vaccines against DENV, a desirable dengue vaccine should elicit robust, long-lasting immunity. Accordingly, length of protection and uses and methods to augment magnitude and duration of CD8+ T cell immunity, if such augmentation is desired, can be obtained by adjusting one or more of the following parameters.
It is widely acknowledged that multiple dosing or higher dosing with replication-incompetent attenuated viruses can induce T cells responses that are comparable to those induced by replication-competent virulent poxviruses (Earl, et al. Nature 428:182-185 (2004); Peters, et al. Vaccine 25:2120-2127 (2007). Based on these observations, in the invention, the route, dose, number of immunizations can be increased, and intervals between immunization optimized.
In general, activated CD8+ T cells are CD44hi CD62Llow Ki-67+Bcl-2low; effector CD8+ T cells are CD107a+ granzyme B+ perforin+; short-lived effector cells (SLECs) are KLRG1+CD127+, memory precursor effector cells (MPECs) are KLRG1−CD127+; central memory T (TCM) are CD62L+CD127±; and effector memory T (TEM) are CD62L−CD127+. It is expected that the highest DENV2E dose (translating to a greater antigenic load over time) and s.c. route (likely leading to the induction of TRM in the skin and perhaps liver in addition to TCM cells) will induce more memory CD8+ T cells than lower doses of DENV2E by way of i.p. adminstration—the greater CD8+ T cell response should respond faster upon viral challenge and correlate with better protection (i.e. the immunized mice should have increased survival and decreased levels of viral RNA in the liver and cytokines in the serum upon viral challenge).
Days between immunization can be optimized, for example, if 30 days between immunizations is too short due to delayed T cell contraction upon repeated immunizations, longer intervals between immunizations, such as 45, 60, or 90 days can be employed.
Finally, the data disclosed herein show that CD4+ T cells are not necessary for CD8+ T cell-dependent protection provided by DENV2E immunization (
This example includes a description of various materials and methods.
C57BL/6 (H-2b) mice were obtained from The Jackson Laboratory and subsequently bred. IFN-α/βR−/− mice on the C57BL/6 background were obtained from Dr. Wayne Yokoyama (Washington University, St. Louis, Mo.) via Dr. Carl Ware. HLA-A*0201/Kb, A*1101/Kb, A*0101, B*0702 and DRB1*0101 transgenic mice were bred at LIAI as previously described (Kotturi et al., Immunome Res 6:4 (2010); Pasquetto et al., J Immunol 175:5504 (2005); Alexander et al., J Immunol 159:4753 (1997); Alexander et al., Hum Immunol 64:211 (2003)). All transgenic mouse strains were subsequently backcrossed with the IFN-α/βR−/− mice at the animal facility at LIAI.B6.SJL mice were purchased from Taconic. Mice were used between 5 and 10 weeks of age.
Mice were infected intravenously (i.v.) in the lateral tail vein or retro-orbitally (r.o.) with 200 μl of the DENV2 strain, S221, in 5% FBS/PBS. Blood was obtained from anesthetized mice by r.o. puncture. For studies with transgenic mice, mice were infected i.v.r.o. with 1010 genomic equivalents (GE) of S221 in 100 uL PBS. On day 7 post-infection, mice were sacrificed and splenic CD8+ or CD4+ T cells, respectively, were used in mouse IFNγ ELISPOT assays. All mouse studies were approved by the Animal Care Committee.
The hybridoma clones SFR3, GK1.5, and 2.43, which produce rat anti-human HLA-DRS, anti-mouse CD4, and anti-mouse CD8 IgG2b Ab, respectively, were from the American Type Culture Collection, and were grown in Protein-Free Hybridoma Medium supplemented with penicillin, streptomycin, HEPES, G1utaMAX, and 2-ME (all from Invitrogen) at 37° C., 5% CO2. C6/36, an A. albopictus mosquito cell line, was cultured in Leibovitz's L-15 Medium (Invitrogen) supplemented with 10% FBS (Gemini Bio-Products), penicillin, streptomycin, and HEPES at 28° C. in the absence of CO2. S221, a plaque-purified DENV2 strain, was derived from the clinical isolate, PL046 (Lin et al., J Virol 72:9729 (1998)), as described previously (Yauch et al., J Immunol 182:4865 (2009)). Viral stocks were amplified in C6/36 cells and purified over a sucrose gradient as previously described (Prestwood et al., J Virol 82:8411 (2008)). Infectious doses were determined based on GE, which were quantified by real-time RT-PCR. There are approximately 5×104 GE/PFU for S221, based on plaque assay on baby hamster kidney cells.
Candidate epitopes were identified using a consensus approach (Wang et al., PLoS Comput Biol 4:e1000048 (2008)). Briefly, all 15-mer peptides that are encoded in the DENV2 PL046 polyprotein were predicted for binding to H-2 I-Ab. Two independent algorithms (Zhang et al., Nucleic Acids Res 36:W513 (2008)) were used to rank the peptides by predicted binding affinity. The median of the two ranks was used to select the top 73 out of 3383 peptides, corresponding to the top 2% of all peptides.
For human MHC class I binding predictions all 9 and 10 mer peptides were predicted for their binding affinity to their respective alleles. Binding predictions were performed using the command-line version of the consensus prediction tool available on the IEDB web site (Zhang et al., Nucleic Acids Res 36:W513 (2008)). Peptides were selected if they are in the top 1% of binders in a given strain. For human MHC class II binding predictions all 15 mer peptides were predicted for their binding affinity to the DRB1*0101 allele. As with class I, binding predictions were performed using the command-line version of the consensus prediction tool available on the IEDB web site. The top 2% of predicted binders were then selected for synthesis. All peptides evaluated in this study were derived from the DENV2 virus strain S221, which was also used as infectious agent in this study, as described above. For the conservancy analysis, full-length DENV polyprotein sequences were retrieved for each serotype from the NCBI Protein database using the following query: txid11053 AND polyprotein AND 3000:5000[slen]. The number of isolates from any one country was limited to 10 to eliminate geographical bias. Sequences were considered “unique” if they varied by at least 1 amino acid from all other sequences. In summary, 171 DENV2, 162 DENV1, 169 DENV3 and 53 DENV4 sequences from the NCBI protein database were investigated for conservancy of the identified epitopes within the respective serotypes.
Peptides utilized in initial screening studies were synthesized as crude material by A and A Labs. A total of 73 15-mer peptides were ordered and synthesized twice in different (alphabetical vs. predicted IC50) order. Positive peptides were re-synthesized by A and A Labs and purified to >90% homogeneity by reverse-phase HPLC. Purity of these peptides was determined using mass spectrometry. The HPLC-purified peptides were used for all subsequent studies.
All peptides using human MHC class I or II sequences were synthesized by Mimotopes (Victoria, Australia). MHC class I predictions led to the synthesis of a total of 431 9-mer and 10-mer peptides. Peptides were made as crude material and combined into pools of 10 individual peptides, according to their predicted HLA restriction. MHC class II predictions resulted in the synthesis of 12 15-mers, which were tested individually.
For surface staining of germinal center B cells, splenocytes were stained with anti-B220Alexa Fluor 647 (Biolegend), anti-CD4-PerCP (BD Biosciences), GL7-FITC (BD Biosciences), anti-IgD-eFluor 450 (eBioscience), and anti-Fas-PE (BD Biosciences). For intracellular cytokine staining (ICS) of CD4+ T cells, 2×106splenocytes were plated in 96-well U-bottom plates and stimulated with individual DENV2 peptides (3 μg/ml) for 2 h (hours). Brefeldin A (GolgiPlug, BD Biosciences) was then added and cells were incubated for another 5 h (hours). Cells were washed, incubated with supernatant from 2.4G2-producing hybridoma cells, and labeled with anti-CD4-eFluor 450 (eBioscience) and anti-CD8α-PerCP-eFluor 710 (eBioscience) or PE-Cy7 (BD Biosciences). The cells were then fixed and permeabilized using the BD Cytofix/Cytoperm Kit, and stained with various combinations of anti-IFN-γ-APC (eBioscience), anti-TNF-PE-Cy7 (BD Biosciences), anti-IL-2-Alexa Fluor 488 (BD Biosciences) or -PE (Biolegend), and anti-CD40L-PE (eBioscience). Foxp3 staining was done using the mouse regulatory T cell staining kit from eBioscience. The criteria for positivity in CD4+ T cell epitope identification were: 2× the percentage of IFN-γ produced by stimulated cells compared with unstimulated cells, positive in two independent crude peptide orders, and positive when ordered as HPLC-purified (>90% pure). For CD8+ T cell ICS, splenocytes (2×106) were stimulated in 96-well U-bottom plates for 5 h (hours) in the presence of 1 μg/ml H-2b-restricted epitopes identified previously: M60-67, NS2A8-15, and NS4B99-107 (Yauch et al., J Immunol 182:4865 (2009)). Anti-CD107a-FITC (BD Biosciences) was added to the wells during the stimulation. Cells were then stained as described for CD4+ T cell ICS. Samples were read on an LSR II (BD Biosciences) and analyzed using FloJo software (Tree Star).
Tissues were embedded in O.C.T. compound (Sakura). Sections (6 μm) were cut and stored at −80° C. Frozen sections were thawed and fixed for 10 minutes in acetone at 25° C., followed by 8 minutes in 1% paraformaldehyde (EMS) in 100 mM dibasic sodium phosphate containing 60 mM lysine and 7 mM sodium periodate pH 7.4 at 4° C. Sections were blocked first using the Avidin/Biotin Blocking Kit (Vector Labs) followed by 5% normal goat serum (Invitrogen) and 1% BSA (Sigma) in PBS. Sections were stained overnight with anti-F4/80-biotin (clone BM8, Biolegend), anti-CD4-PE (clone RM4-5, eBioscience), anti-CD8β-Alexa Fluor 647 (clone YTS156.7.7, Biolegend), and anti-B220-FITC (clone RA3-6B2, BD Pharmingen). Sections were then washed and stained with streptavidin-Alexa Fluor 750 and rabbit anti-FITC-Alexa Fluor 488 (Invitrogen). Images were recorded using a Leica TCS SP5 confocal microscope, processed using Leica Microsystems software, stitched together using Adobe Illustrator, and adjusted using ImageJ.
Hybridoma supernatants were clarified by centrifugation, dialyzed against PBS, sterile-filtered, and quantified by BCA Protein Assay Reagent (Thermo Scientific), IFN-α/βR−/− mice were injected i.p. with 250 μg of SFR3, or GK1.5, or 2.43 in PBS (250 μl total volume) 3 days and 1 day before or 1 day before and 1 day after infection, which resulted in depletion of ≧90% of CD8+ cells and ≧97% of CD4+ cells. In
Serum was harvested from control and CD4-depleted IFN-α/βR−/− mice 7 days after infection with 1010 GE of DENV2, or naïve mice. EIA/RIA 96-well plates (Costar) were coated with DENV2 (109 GE per well) in 50 μl 0.1M NaHCO3. The virus was UV-inactivated and plates left overnight at 4° C. The plates were then washed to remove unbound virus using 0.05% (v/v) Tween 20 (Sigma) in PBS. After blocking with Blocker Casein Blocking Buffer (Thermo Scientific) for 1 h at room temperature, 1:3 serial dilutions of serum in a total volume of 100 μl were added to the wells. After 1.5 h, wells were washed and bound antibody was detected using HRP-conjugated goat anti-mouse IgG Fc portion or HRP-conjugated donkey anti-mouse IgMμ chain (Jackson Immunoresearch) and TMB (eBioscience).
Serum was heat-inactivated at 56° C. for 30 min. Three-fold serial dilutions of serum were then incubated with 5×108 GE of DENV2 for 1 h at room temperature in a total volume of 100 μl PBS. Next, approximately 6×105 C6/36 cells per well of a 24-well plate were infected with 100 μl of the virus-antibody mix for one hour at 28° C. Cells were washed twice with 500 μl of PBS, and then incubated at 28° C. in 500 μl L-15 Medium containing 5% FBS, penicillin, and streptomycin for 24 h. For each antibody dilution, the percentage of infected cells was determined by flow cytometry as previously described (Lambeth et al., J Clin Microbiol 43:3267 (2005)) using 2H2-biotin (IgG2a anti-prM/M, DENV1-4 reactive) and streptavidin-APC (Biolegend). The percentage of infected cells was normalized to 100% (infection without serum).
IFN-α/βR−/− mice (recipients) were infected with 1010 GE of DENV2. Some mice were depleted of CD4+ T cells before infection. Splenocytes (targets) were harvested from donor B6.SJL congenic mice (CD45.1) 7 days later. RBC were lysed, and the target cells were pulsed with varying concentrations of a pool of 4 H-2b-restricted DENV2 peptides (M60-67, NS2A8-15, NS4B99-107, NS5237-245) or DMSO for 1 h at 37° C. The cells were then washed and labeled with CFSE (Invitrogen) in PBS/0.1% BSA for 10 min at 37° C. Cells were labeled with 1 μM CFSE (CFSEhigh)or 100 nM CFSE (CFSElow) or left unlabeled. After washing, the cell populations were mixed and 5×106 cells from each population were injected i.v.into naïve or infected recipient mice. After 4 h, the mice were sacrificed and splenocytes stained with anti-CD45.1-APC (eBioscience) and analyzed by flow cytometry, gating on CD45.1+ cells. The percentage killing was calculated as follows: 100−((percentage DENV peptide-pulsed in infected mice/percentage DMSO-pulsed in infected mice)/(percentage DENV peptide-pulsed in naïve mice/percentage DMSO-pulsed in naïve mice)×100).
IFN-α/βR−/− mice (recipients) were infected with 1010 GE of DENV2. Some mice were depleted of CD4+ or CD8+ cells before infection. Splenocytes (targets) were harvested from donor B6.SJL congenic mice (CD45.1) 7 days later. RBC were lysed and the target cells were pulsed with 1.7 μg (approximately 1 μM) each of NS2B108-122, NS3198-212, and NS3237-251 (or DMSO) for 1 h at 37° C. The cells were then washed and labeled with CFSE in PBS/0.1% BSA for 10 min at 37° C. DENV2 peptide-pulsed cells were labeled with 1 μM CFSE (CFSEhigh) and DMSO-pulsed cells with 100 nM CFSE (CFSElow). After washing, the two cell populations were mixed and 5×106 cells from each population were injected i.v. into naïve or infected recipient mice. After 16 h, the mice were sacrificed and splenocytes stained and the percentage killing calculated as described for the CD8 in vivo cytotoxicity assay.
Mice were euthanized by isoflurane inhalation and blood was collected via cardiac puncture. Serum was separated from whole blood by centrifugation in serum separator tubes (Starsted). Small intestines were put into PBS, flushed, filleted, chopped into small pieces, and put into RNA later (Qiagen). Other organs were immediately placed into RNAlater and all organs were subsequently homogenized for 3 min in 1 ml tissue lysis buffer (Qiagen Buffer RLT) using a Mini-Beadbeater-8 (BioSpec Products) or QiagenTissueLyser. Immediately after homogenization, all tissues were centrifuged (5 min, 4° C., 16,000×g) to pellet debris, and RNA was isolated using the RNeasy Mini Kit (Qiagen). Serum RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen). After elution, viral RNA was stored at −80° C. Quantitative RT-PCR was performed according to a published protocol (Houng et al., J Virol Methods 86:1-11 (2000)), except a MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) with One-Step qRT-PCR Kit (Quanta BioSciences) were used. The DENV2 standard curve was generated with serial dilutions of a known concentration of DENV2 genomic RNA which was in vitro transcribed (MAXlscriptKit, Ambion) from a plasmid containing the cDNA template of S221 3′UTR. After transcription, DNA was digested with DNase I, and RNA was purified using the RNeasy Mini Kit and quantified by spectrophotometry. To control for RNA quality and quantity when measuring DENV in tissues, the level of 18S rRNA was measured using 18S primers described previously (Lacher, et al., Cancer Res 66:1648 (2006)) in parallel real-time RT-PCR reactions. A relative 18S standard curve was made from total splenic RNA.
IFN-α/βR−/− mice were immunized s.c. with 50 μg each of NS2B108-122, NS3198-212, and NS3237-251 emulsified in CFA (Difco). After 11 days, mice were boosted with 50 μg peptide emulsified in IFA (Difco). Mock-immunized mice received PBS/DMSO emulsified in CFA or IFA. Mice were infected 13 days after the boost with 1011 GE of DENV2 (some mice were depleted of CD4+ or CD8+ T cells 3 days and 1 day before infection). Four days later, the mice were sacrificed and tissues harvested, RNA isolated, and DENV2 RNA levels measured as described above. For Example 7, mice were immunized instead with 50 μg each of C51-59, NS2A8-15, NS4B99-107, and NS5237-245 as described in Yauch et al., J Immunol 182:4865 (2009).
MHC purification and quantitative assays to measure the binding affinity of peptides to purified A*0201, A*0101, A*1101, B*0702 and DRB1*0101 molecules were performed as described elsewhere(Sidney et al., Immunome Res 4:2 (2008); Sidney et al., Curr Protoc Immunol Chapter 18:Unit 18 13 (2001)). Briefly, after a 2-day incubation, binding of the radiolabeled peptide to the corresponding MHC molecule was determined by capturing MHC/peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-One, Monroe, N.C.) coated with either the W6/32 (HLA class I specific) or L243 (HLA DR specific) monoclonal antibodies. Bound cpmwere then measured using the Top count microscintillation counter (Packard Instrument, Meriden, Conn.). The concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide (IC50) was then calculated.
The tumor cell line 721.221(Shimizu et al., J Immunol 142:3320 (1989), which lacks expression of HLA-A, -B and C class I genes, was transfected with the HLA-A*0201/Kb or HL-A*1101 chimeric genes, and was used as APC in the restriction assays. The non-transfected cell line was used as a negative control.
Peripheral blood samples were obtained from healthy adult blood donors from the National Blood Center in Colombo, Sri Lanka. PBMC were purified by density gradient centrifugation (Ficoll-Hypaque, Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions. Cell were suspended in fetal bovine serum (Gemini Bio-products, Sacramento, Calif.) containing 10% dimethyl sulfoxide, and cryo-preserved in liquid nitrogen. DENV seropositivity was determined by ELISA. A flow cytometry-based neutralization assays was performed for further characterization of seropositve donors, as previously described (Kraus et al., J Clin Microbiol 45:3777 (2007)).
Genomic DNA isolated from PBMC of the study subjects by standard techniques (QIAmp. Qiagen, Valencia, Calif.) was use for HLA typing. High resolution Luminex-based typing for HLA Class I and Class II was utilized according the manufacturer's protocol (Sequence-Specific Oligonucleotides (SSO) typing; One Lambda, Canoga Park, Calif.). Where needed, PCR based methods were used to provide high resolution sub-typing. (Sequence-Specific Primer (SSP) typing; One Lambda, Canoga Park, Calif.).
For all murine studies, splenic CD4+ or CD8+ T cells were isolated by magnetic bead positive selection (MiltenyiBiotec, BergischGladbach, Germany) 7 days after infection. 2 ×105 T cells were stimulated with 1×105 uninfected splenocytes as APCs and 10 μg/ml of individual DENV peptides in 96-well flat-bottom plates (Immobilon-P; Millipore, Bedford, Mass.) coated with anti-IFNγ mAb (clone AN18; Mabtech, Stockholm, Sweden). Each peptide was evaluated in triplicate. Following a 20-h incubation at 37° C., the wells were washed with PBS/0.05% Tween 20 and then incubated with biotinylated IFNγ mAb (clone R4-6A2; Mabtech) for 2 h. The spots were developed using Vectastain ABC peroxidase (Vector Laboratories, Burlingame, Calif.) and 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, Mo.) and counted by computer-assisted image analysis (KS-ELISPOT reader, Zeiss, Munich, Germany). Responses against peptides were considered positive if the net spot-forming cells (SFC) per 106 were ≧20, a stimulation index of ≧2, and p<0.05 in a t test comparing replicates with those from the negative control.
To evaluate the antigenicity of the epitopes in humans, 2×106 PBMC/ml were stimulated in the presence of 1 μg/ml individual peptide for 7 days. Cells were cultured at 37° C., 5% CO2, and recombinant IL2 (10 U/mL, eBiosciences, San Diego, Calif.) was added 3 days after antigenic stimulation. After one week, PBMC were harvested and tested at a concentration of 1×105/well in an IFNγ ELISPOT assay, as described above. The mAb 1-D1K and mAb 7-B6-1 (Mabtech) were used as coating and biotinylated secondary Ab, respectively. To be considered positive, IFNγ responses needed to exceed the threshold set as the mean responses of HLA non-matched and DENV seronegative donors plus 3 times the standard deviation.
Data were analyzed with Prism software version 5.0 (GraphPad Software, Inc.). Statistical significance was determined using the unpaired t-test with Welch's correction.
This example includes data demonstrating CD4+ T cell activation and expansion following DENV2 infection.
DENV2 (1010 GE of S221) infection of IFN-α/βR−/− mice results in an acute infection, with viral replication peaking between 2 and 4 days after infection (Yauch, et al. J Immunol 182:4865 (2009)). At this time the mice show signs of disease including hunched posture and ruffled fur, and the virus is subsequently cleared from the serum by day 6. To determine the role of CD4+ T cells during the course of this primary DENV2 infection, the expansion of CD4+ T cells in the spleens of IFN-α/βR−/− mice 7 days after infection with DENV2 was examined, and a 2-fold increase in CD4+ T cell numbers was observed (
Regulatory T cells (Tregs) are a subset of CD4+ T cells that are characterized by the expression of the transcription factor, Foxp3 (Josefowicz, et al. Immunity 30:616 (2009)), and have been found to facilitate the early host response to HSV-2 (Lund, et al. Science 320:1220 (2008)) and help control WNV infection (Lanteri, et al. J Clin Invest 119:3266 (2009)). To determine if DENV2 infection resulted in an expansion of Tregs, the number of CD4+Foxp3+ cells in the spleen 7 days after infection was determined. There was a decrease in the percentage of Tregs among total CD4+ cells, and no change in the number of Tregs, demonstrating that DENV2 infection does not lead to an expansion of Tregs in the spleen (
This example includes data for the identification of DENV2 CD4+ T cell epitopes and phenotype of DENV2-specific CD4+ T cells.
In order to study the DENV2-specific CD4+ T cell response, the identity of MHC class II (I-Ab)-restricted CD4+ T cell epitopes using a bioinformatics prediction method previously reported to map the CD4+ T cell response to mouse cytomegalovirus (Arens, et al. J Immunol 180:6472 (2008)) was employed. Briefly, the proteome of DENV2 was screened and 73 15-mer peptides predicted to bind I-Ab were identified. The peptides were tested by IFN-γ ICS using splenocytes from DENV2-infected IFN-α/βR−/−mice. Positive peptides (2× background) were then re-ordered as HPLC-purified (>90%) and re-tested. Four positive peptides were identified: NS2B108-122, N53198-212, N53237-251, and NS4B96-110 (
Multicolor flow cytometry was performed to study the phenotype of DENV2-specific CD4+ T cells. These cells produced IFN-γ, TNF, and IL-2 (
This example includes a description of studies of the effects of CD4+ and/or CD8+ T cell depletions on DENV2 viral RNA levels, and data showing that CD4+ T cells are not required for the anti-DENV2 antibody response, and are also not necessary for the primary DENV2-specific CD8+ T cell response.
To determine how CD4+ T cells contribute to controlling DENV2 infection, CD4+ T cells, CD8+ T cells, or both were depleted from IFN-α/βR−/− mice and DENV2 RNA levels 5 days after infection with 1010 GE of DENV2 was measured. No difference in viral RNA levels between control undepleted mice and CD4-depleted mice in the serum, kidney, small intestine, spleen, or brain was observed (
Although CD4+ T cells were not required for controlling DENV2 infection, the contribution to the anti-DENV immune response, for example by helping the B cell and/or CD8+ T cell responses, was investigated. CSR, the process by which the immunoglobulin heavy chain constant region is switched so the B cell expresses a new isotype of Ab, can be induced when CD40L-expressing CD4+ T cells engage CD40 on B cells (Stavnezer, et al. Annu Rev Immunol 26:261 (2008)). However, CSR can also occur in the absence of CD4+ T cell help. To determine whether the anti-DENV2 antibody response depends on CD4+ T cells, DENV2-specific IgM and IgG titers in the sera of control and CD4-depleted mice was measured 7 days after infection with 1010 GE of DENV2. As expected, there was no difference in IgM titers at day 7 between control and CD4-depleted mice (
Next, the role of CD4+ T cells in helping the CD8+ T cell response was assessed by examining the DENV2-specific CD8+ T cell response in control and CD4-depleted DENV2-infected mice. The numbers of splenic CD8+ T cells were equivalent in control and CD4-depleted mice. IFN-γ ICS was performed using DENV2-derived H-2b-restricted immunodominant peptides identified (M60-67, NS2A8-15, and NS4B99-107) (Yauch, et al. J Immunol 182:4865 (2009)). Somewhat surprisingly, there was an increase in the number of DENV2-specific IFN-γ+CD8+ T cells in CD4-depleted mice compared with control mice (
This example is a description of studies of in vivo killing of I-Ab-restricted peptide-pulsed target cells in DENV2-infected mice, and data showing that vaccination with DENV2 CD4+ T cell epitopes controls viral load.
Although the absence of CD4+ T cells had no effect on viral RNA levels on day 5 after infection, it was possible that CD4+ T cells could still be contributing to the anti-DENV2 host response by killing infected cells. In vivo cytotoxicity assay was performed using splenocytes pulsed with the three peptides that contain only CD4+ T cell epitopes (NS2B108-122, NS3198-212, and NS3237-251) and not NS4B96-110 to measure only CD4+, not CD8+ T cell-mediated killing. Approximately 30% killing of target cells was observed (
Having found that DENV2-specific CD4+T cells can kill target cells, immunization with CD4+T cell epitopes was assessed for control of DENV2 infection. Mice were immunized with NS2B108-122, NS3198-212, and NS3237-251 before DENV2 infection, and CD4+ T cell responses by ICS and viral RNA levels 4 days after infection measured. Peptide immunization resulted in enhanced CD4+ T cell cytokine responses, and significantly lower viral loads in the kidney and spleen (
This example includes a discussion of the data and a summary of the implications.
The data reveal that CD8+ T cells play an important protective role in the response to primary DENV2 infection, whereas CD4+ T cells do not. CD4+ T cells expanded, were activated, and were located near CD8+ T cells and B cells in the spleen after DENV2 infection, yet they did not seem to affect the induction of the DENV2-specific CD8+ T cell or antibody responses. In fact, CD4+ T cell depletion had no effect on viral clearance. However, the data demonstrate that vaccination with CD4+ T cell epitopes prior to DENV infection can provide significant protection, demonstrating that T cell peptide vaccination is a strategy for DENV immunization without the risk of ADE.
The DENV2-specific CD4+ T cells recognized epitopes from the NS2B, NS3, and NS4B proteins, and displayed a Th1 phenotype. CD4+ T cell epitopes have been identified in mice infected with other flaviviruses, including YFV, for which an I-Ab-restricted peptide from the E protein was identified (van der Most, et al. Virology 296:117 (2002)), and WNV, for which six epitopes from the E and NS3 proteins were identified (Brien, et al. J Immunol 181:8568 (2008)). DENV-derived epitopes recognized by human CD4+ T cells have been identified, primarily from NS proteins, including the highly conserved NS3 (Mathew, et al. Immunol Rev 225:300 (2008)). One study identified numerous epitopes from the NS3200-324 region, and alignment of consensus sequences for DENV1-4 revealed that this region is more conserved (78%) than NS3 as a whole (68%) (Simmons, et al. J Virol 79:5665 (2005)), suggesting that the region contains good candidates for a DENV T cell epitope-based vaccine. Interestingly, one of the NS3-derived epitopes identified herein is also a human CD4+ T cell epitope, which may bind human HLAs promiscuously, making it a good vaccine candidate. Another finding was that one of the CD4+ T cell epitopes identified in this study contained the most immunodominant of the CD8+ T cell epitopes identified previously. Overlapping epitopes have also been found in LCMV (Homann, et al. Virology 363:113 (2007); Mothe, et al. J Immunol 179:1058-1067 (2007); Dow, et al. J Virol 82:11734 (2008)). The significance of overlapping epitopes is unknown, but is likely related to homology between MHC class I and MHC class II, and may be associated with proteasomal processing. Overlapping epitopes may turn out to be common once the complete CD4+ and CD8+ T cell responses to other pathogens are mapped.
CD4+ T cells are classically defined as helper cells, as they help B cell and CD8+ T cell responses. However, inflammatory stimuli can override the need for CD4+ T cell help, and therefore, the responses to many acute infections are CD4-independent (Bevan, Nat Rev Immunol 4:595 (2004)). DENV2 replicates to high levels in IFN-α/βR−/− mice, the mice appear hunched and ruffled at the time of peak viremia, and they have intestinal inflammation, suggesting that there is a significant inflammatory response to DENV2. Accordingly, CD4+ T cells did not play a critical role in the immune response to primary DENV2 infection. The contribution of CD4+ T cells has been examined during infections with other flaviviruses. The reports suggest that the contribution of CD4+ T cells to protection against flavivirus infection varies depending on the virus and experimental system (Brien, et al. J Immunol 181:8568 (2008); Murali-Krishna, et al. J Gen Virol 77 (Pt 4):705 (1996); Sitati, et al. J Virol. 80:12060 (2006)).
Antibody responses can be T cell-dependent or T cell-independent. In particular, the formation of GCs is thought to be CD4+ T cell-dependent, and is where high-affinity plasma cells and memory B cells are generated and where CSR can occur (Stavnezer, et al. Annu Rev Immunol 26:261 (2008); Allen, et al. Immunity 27:190 (2007); Fagarasan et al. Science 290:89 (2000)). T-independent antibody responses to viruses have been demonstrated for vesicular stomatitis virus (Freer, et al. J Virol 68:3650 (1994)), rotavirus (Franco, et al. Virology 238:169 (1997)), and polyomavirus (Szomolanyi-Tsuda, et al. Virology 280:160 (2001)). In addition, EBV (via LMP1) can induce CD40-independent CSR (He, et al. J Immunol 171:5215 (2003)), and mice deficient for CD40 or CD4+ T cells are able to mount an influenza-specific IgG response that is protective (Lee, et al. J Immunol 175:5827 (2005)).
The data herein demonstrate that the DENV2-specific IgG response at day 7 is CD4-independent. The lack of GC B cells in CD4-depleted mice shows that CD4-depletions have a functional effect, and indicate anti-DENV IgG is being produced by extrafollicular B cells. It is possible that the absence of CD4+ T cells would have an effect on DENV2-specific antibody titers and/or neutralizing activity at later time points, however, the goal of this study was to determine how CD4+ T cells contribute to clearance of primary DENV2 infection, and the early anti-DENV2 antibody response is CD4-independent.
Like pathogen-specific antibody responses, primary CD8+ T cell responses to many acute infections are also CD4-independent. CD4-independent CD8+ T cell responses have been demonstrated for Listeria monocytogenes (Sun, et al. Science 300:339 (2003); Shedlock, et al. J Immunol 170:2053 (2003)), LCMV (Ahmed, et al. J Virol 62:2102 (1988)), and influenza (Belz, et al. J Virol 76:12388 (2002)). Recently a mechanism for how DCs can activate CD8+ T cells in the absence of CD4+ T cell help has been described (Johnson, et al. Immunity 30:218 (2009)). In accordance with the studies herein, the primary CD8+ T cell response to DENV2 did not depend on CD4+ T cells. In fact, an enhanced DENV2-specific CD8+ T cell response in CD4-deficient mice compared with control mice at day 7 was observed, which has also been reported for influenza—(Belz, et al. J Virol 76:12388 (2002)) and WNV—(Sitati, et al. J Virol. 80:12060 (2006)) specific CD8+ T cell responses. This could be due to the depletion of Tregs, or an increased availability of cytokines (e.g. IL-2) in mice lacking CD4+ T cells. This enhanced CD8+ T cell response may explain why CD4-depleted mice have no differences in viral titers despite the fact that DENV2-specific CD4+ T cells demonstrate in vivo cytotoxicity.
Although CD4+ T cells did not play an important role in helping antibody or CD8+ T cell responses, DENV2-specific CD4+ T cells could kill peptide-pulsed target cells in vivo. CD4+ T cells specific for other pathogens, including HIV (Norris, et al. J Virol 78:8844 (2004)) and influenza (Taylor, et al. Immunol Lett 46:67 (1995)) demonstrate in vitro cytotoxicity. In vivo cytotoxicity assays have been used to show CD4+ T cell-mediated killing following infection with LCMV (Jellison, et al. J Immunol 174:614 (2005)) and WNV (Brien, et al. J Immunol 181:8568 (2008)). DENV-specific cytolytic human CD4+ T cell clones (Gagnon, et al. J Virol 70:141 (1996); Kurane, et al. J Exp Med 170:763 (1989)) and a mouse (H-2d) CD4+ T cell clone (Rothman, et al. J Virol 70:6540 (1996)) have been reported. Whether CD4+ T cells actually kill infected cells during DENV infection remains to be determined, but is possible, as MHC class II-expressing macrophages are targets of DENV infection (Zellweger, et al. Cell Host Microbe 7:128 (2010)). Based on the fact that CD4-depletion did not have a significant effect on viral clearance, it is unlikely that CD4+ T cell-mediated killing plays a major role in the anti-DENV2 response in this model.
A caveat to using the IFN-α/IβR−/− mice is that type I IFNs are known to help T cell and B cell responses through their actions on DCs, and can act directly on T cells (Iwasaki, et al. Nat Immunol 5:987 (2004)). Type I IFNs were found to contribute to the expansion of CD4+ T cells following infection with LCMV, but not Listeria monocytogenes (Havenar-Daughton, et al. J Immunol 176:3315 (2006)). Type I IFNs can induce the development of Th1 IFN-γ responses in human CD4+ T cells, but cannot substitute for IL-12 in promoting Th1 responses in mouse CD4+ T cells (Rogge, et al. J Immunol 161:6567 (1998)). Following Listeria infection, IL-12 synergized with type I IFN to induce IFN-γ production by CD4+ T cells (Way, et al. J Immunol 178:4498 (2007)). Although DENV does not replicate to detectable levels in wild-type mice, examining the CD4+ T cell response in these mice revealed that the same epitopes were recognized as in the IFN-α/βR−/− mice, but the magnitude of the epitope-specific response was greater in the IFN-α/βR−/− mice. This suggests that the high levels of viral replication in the IFN-α/βR−/− mice are sufficient to drive a DENV2-specific CD4+ IFN-γ response. The results demonstrate a DENV2-specific CD4+ T cell response, including Th1-type cytokine production and cytotoxicity, in the absence of IFN-α/βR signaling; however, this response is not required for clearance of infection. It is possible that CD4+ T cells contribute to protection during DENV infection of hosts with intact IFN responses.
The results herein demonstrate that immunization with CD4+ T epitopes is also protective. These results have significant implications for DENV vaccine development, since designing a vaccine is challenging, as, ideally, a vaccine needs to protect against all four serotypes. DENV vaccine candidates in development, some of which are in phase II trials, focus on eliciting an antibody response. The challenge is to induce and maintain a robust neutralizing antibody response against all four serotypes, as it is becoming increasingly clear that non-neutralizing antibodies (or sub-neutralizing quantities of antibodies) can actually worsen dengue disease (Zellweger, et al. Cell Host Microbe 7:128 (2010); Balsitis, et al. PLoS Pathog 6:e1000790 (2010)). An alternative approach would be a peptide vaccine that induces cell-mediated immunity, including both CD4+ and CD8+ T cell responses, which, although not able to prevent infection, would reduce viral loads and disease severity, and would eliminate the risk of ADE. Such a vaccine should target highly conserved regions of the proteome, for example NS3, NS4B, and/or NS5, and ideally include epitopes conserved across all four serotypes. A vaccine containing only peptides from these particular NS proteins would also preclude the induction of any antibody against epitopes on the virion, which could enhance infection, or antibody against NS1, which could potentially contribute to pathogenesis (Lin, et al. Viral Immunol 19:127 (2006)). Peptide vaccination was given along with CFA, which is commonly used in mice to induce Th1 responses (Billiau, et al. J Leukoc Biol 70:849 (2001)), which was the type of response observed after natural DENV infection. CFA is not a vaccine adjuvant approved for human use, and thus, any peptide vaccine developed against DENV will be formulated with an adjuvant that is approved for human use.
Although the results herein indicate that CD4+ T cells do not make a significant contribution to controlling primary DENV2 infection, the characterization of the primary CD4+ T cell response and epitope identification allows the determination of the role of CD4+ T cells during secondary homologous and heterologous infections. CD4+ T cells are often dispensable for the primary CD8+ T cell response to infection, but have been shown to be required for the maintenance of memory CD8+ T cell responses after acute infection (Sun, et al. Nat Immunol 5:927 (2004)). Finally, the data herein support a DENV vaccine strategy that induces CD4+ T cell, in addition to CD8+ T cell, responses.
This example includes a description of additional studies showing that vaccination with DENV CD8+ T cell epitopes controls viral load.
Since depleting CD8+ T cells resulted in increased viral loads and DENV-specific CD8+ T cells demonstrated in vivo cytotoxic activity, studies were performed to determine whether enhancing the anti-DENV CD8+ T cell response through peptide immunization would contribute to protection against a subsequent DENV challenge. Specifically, the effect of peptide vaccination on viremia was determined by immunizing IFN-α/βR−/− mice with DENV-2 derived H-2b peptides prior to infection with S221. Mice were immunized with four dominant DENV epitopes (C51-59, NS2A8-15, NS4B99-107, and N55237-245) (Yauch et al., J Immunol 182:4865 (2009)) in an attempt to induce a multispecific T cell response, which is desirable to prevent possible viral escape through mutation (Welsh et al., Nat Rev Microbiol 5:555 (2007)). At day 4 after infection, viremia in the serum was measured by real-time RT-PCR, as described above. The peptide-immunization resulted in enhanced control of DENV infection, with 350-fold lower serum DENV RNA levels in peptide-immunized mice than mock-immunized mice (Yauch et al., J Immunol 182:4865 (2009)). To confirm that the protection was mediated by CD8+ T cells, CD8+ T cells were depleted from a group of peptide-immunized mice prior to infection, and it was found that this abrogated the protective effect (Yauch et al., J Immunol 182:4865 (2009)). Thus, the data demonstrate that a preexisting DENV-specific CD8+ T cell response induced by peptide vaccination enhances viral clearance.
Most dengue infections are asymptomatic or classified as DF, whereas DHF/DSS accounts for a small percentage of dengue cases, indicating that in most infections the host immune response is protective. These data indicate that CD8+ T cells contribute to protection during primary infection by reducing viral load and that CD8+ T cells are an important component to a protective immune response.
This study shows that immunization with four dominant epitopes prior to infection resulted in enhanced DENV clearance, and this protection was mediated by CD8+ T cells. These results indicate that vaccination with T cell epitopes can reduce viremia.
Results from the Examples described herein reveal a critical role for CD8+ T cells in the immune response to an important human pathogen, and provide a rationale for the inclusion of CD8+ T cell epitopes in DENV vaccines. Furthermore, identification of the CD8+ T cell epitopes recognized during DENV infection in combination with the disclosed mouse model can provide the foundation for elucidating the protective versus pathogenic role of CD8+ T cells during secondary infections.
This example is a description of a novel system to identify DENV specific HLA*0201 epitopes.
Mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR−/− mice. HLA*0201 transgenic and IFN-α/βR−/− mice strains were backcrossed to study DENV-specific HLA restricted T cell responses. These mice were then infected with mouse adapted DENV2 strain S221, and purified splenic T cells were used to study the anti-DENV CD8+ T cell responses.
A panel of 116 predicted A*0201 binding peptides were generated using bioinformatics (Moutaftsi, et al. Nat Biotechnol 24:817 (2006)). Predicted HLA A*0201 binding peptides were combined into pools of 10 individual peptides and tested in an IFNγ ELISPOT assay using CD8+ T cells from HLA transgenic IFN-α/⊕R−/− and IFN-α/βR+/+, S221 infected mice, respectively. Positive pools were deconvoluted and the individual peptides were tested in two independent studies. Using this approach, a single peptide in the HLA*A0201 IFN-α/βR+/+ mice was identified (NS53058-3066,
This example describes population coverage by additional HLA transgenic mice IFN-α/βR−/− strains.
To address whether similar observations could be made by assessing responses in other HLA-transgenic IFN-α/βR−/− and IFN-α/βR−/− mice, IFN-α/βR−/− mice were backcrossed with HLA A*0101, A*1101, and B*0702 transgenic mice. These alleles were chosen as representatives of three additional HLA class I supertypes (A1, A3 and B7, respectively).
Screening in HLA A*0101 and A*1101 transgenic IFN-α/βR−/− mice revealed 9 HLA A*0101 restricted (
To extend the observations to mice transgenic for an HLA B allele HLA B*0702 transgenic IFN-α/βR−/− and IFN-α/βR+/+ mice were infected and epitope recognition was compared between the two strains. 15 B*0702 restricted epitopes in the IFN-α/βR−/− strain (
This example describes Dengue virus specific T cell responses in an MHC class II transgenic mouse model.
To determine if the observations made in the case of MHC class I transgenic mice were also applicable to MHC class II molecules, the antigenicity of HLA DRB1*0101 DENV predicted binding peptides in HLA DRB1*0101, IFN-α/βR−/− and IFN-α/βR+/+ mice, respectively, was determined. Using the same study conditions described above for the MHC class I transgenic mice, HLA DRB1*0101, IFN-α/βR−/− and IFN-α/βR+/+ mice were infected with DENV2 (S221), and CD4+ T cells were isolated 7 days post infection. A panel of 12 predicted S221 specific peptides was then analyzed for IFNγ production by ELISPOT. Five epitopes in the DRB1*0101, IFN-α/βR−/− mice were identified from these assays which could elicit a significant IFNγ response in two independent studies (
In summary, a total of 55 epitopes were identified in the HLA transgenic IFN-α/βR−/− mice, whereas the same screen in HLA transgenic IFN-α/βR+/+ mice only revealed 8 epitopes. All of these 8 epitopes have also been detected in the HLA transgenic IFN-α/βR−/− mice. The broader repertoire seen in IFN-α/βR−/− mice as well as the stronger and more robust IFNγ responses, suggest that HLA transgenic mice, backcrossed with IFN-α/βR−/− mice are a more suitable model to study T cell responses to DENV infection than HLA transgenic wildtype mice.
This example is a description of mapping optimal epitopes with respect to peptide length, and further characterization of the identified epitopes.
For all HLA alleles tested in this study, class I 9- and 10-mer peptide predictions were performed using the consensus prediction tool as described in greater detail in Example 1. Within the 50 MHC class I restricted epitopes identified, 9 pairs of nested epitopes were identified, where the 9-mer as well as the 10-mer peptide was able to elicit an immune response. To determine which specific peptide within each nested epitope pair was the optimal epitope, peptide titration assays were employed (
Similarly, for two of the identified B*0702 restricted epitopes (NS4B2296-2305 and NS52646-2655) which showed low binding affinity (IC50>1000 nM) 8-, and 9-mers carrying alternative dominant B7 motifs were synthesized and tested them for T cell recognition and binding affinity. In one case the corresponding 8-mer (NS4B2296-2304) showed dominant IFNγ responses as well as higher binding affinity compared to the 9-mer. In the other case, the l0 mer originally identified (NS52646-2655) was able to elicit higher responses than the newly synthesized 8- and 9-mer. In both cases the optimal epitope length could be identified and was considered further in the study, as shown in
Of all five HLA transgenic mouse strains analyzed, two strains, namely the A*0201 and the A*1101 transgenic strains, co-expressed murine MHC molecules together with the respective HLA molecule. Thus it was necessary to address that the observed responses were restricted by the human HLA class I molecule and not by murine Class I. Accordingly, purified T cells were studied for their capacity to recognize the specific epitopes when pulsed on antigen presenting cells expressing only human HLA class and not any murine class I molecule. For this purpose, the tumor cell line 721.221 was utilized, which is negative for expression of any human or murine Class I molecule, and was transfected with either HLA A*0201 or HLA*1101.
As shown in
To further confirm the MHC restriction of the identified epitopes, MHC-binding capacity to their predicted allelic molecule was measured using purified HLA molecules in an in vitro binding assay. The results of these assays are also shown in Table 2. 32 of the 42 tested peptides (67%) bound the corresponding predicted allele with high affinity as indicated by an IC50<50 nM. 16 out of these even showed an IC50<10 nM and can therefore be considered as very strong binders. Of the remaining peptides, 7 (17%) were able to bind the predicted allele with intermediate affinities as indicated by IC50<150 nM. Only three of the identified epitopes (7%) bound with low affinity, showing an IC50>500nM. A summary of all epitopes identified, after conclusion of the studies and elimination of redundancies, is shown in Table 2.
1[76] Simmons et al., J Virol 79:5665 (2005)
2[77] Appanna et al,. Clin Vaccine Immunol 14:969 (2007)
3[78] Mongkolsapaya et al., J Immunol 176:3821
4[79] Wen et al., Virus Res 132:42 (2008)
This example includes a description of validation studies of the identified epitopes in human DENV seropositive donors.
To validate the epitopes identified in the HLA-transgenic IFN-α/βR−/− mice, the capacity of these epitopes to stimulate PBMC from human donors, previously exposed to DENV, was analyzed. Since the IFNγ response to these peptides was not detectable ex vivo, HLA-matched PBMC were re-stimulated for 7 days in presence of the respective peptides and IL2. As a control PBMC from donors which neither expressed the exact HLA-molecule nor one from the same supertype, as well as PBMC from DENV seronegative donors were re-stimulated. The average IFNγ response from these donors plus 3 times the standard deviation (SD) was set as a threshold of positivity.
This example includes studies showing dominance of B7 responses.
A notable observation here was that out of all HLA transgenic mouse strains tested the strongest CD8+ T cell responses could be detected in the B*0702 transgenic IFN-α/βR−/− mice. Four B*0702 restricted epitopes were able to elicit an IFNγ response above a thousand SFC/106 CD8+ T cells. On average B*0702 epitopes were able to elicit an IFNγ response of 857 SFC/106 CD8+ T cells, compared to an average of 350, 365, and 476 SFC/106 CD8+ T cells for the HLA A*0101, A*0201 and A*1101 restricted epitopes, respectively (
This example includes a description of studies showing the subprotein location of identified epitopes, and the conservancy of identified epitopes within the DENV2 serotype.
The identified epitopes are derived from 9 of the 10 DENV proteins, with the membrane protein being the only protein where no epitope could be detected (
Cross-reactivity of T cells is a well-described phenomenon in DENV infection (Mathew, et al. Immunol Rev 225:300 (2008))). To circumvent this issue, T cell reactivity was exclusively tested to S221 derived peptides, which was also used as infectious agent in this study. However, to assess the relevance for infections with other DENV2 strains, conservancy of these epitopes within the DENV2 serotype was analyzed. 171 full-length DENV2 polyprotein sequences from the NCBI
Protein database were analyzed for conservancy. Of the epitopes identified, 30 out of the 42 epitopes were conserved in >90% of all DENV2 strains; 8 epitopes were even conserved in all 171 strains analyzed. Of the remaining 12 epitopes, 6 were conserved in >75% of all strains analyzed and the other half was found in the 30-65% range. This accounts for an average conservancy of 92% for the epitopes identified, which is significantly higher than the average conservancy of non-epitopes (73%; p<0.001).
To determine if the epitopes identified were also conserved in serotypes, other than DENV2, 162 DENV1, 169 DENV3 and 53 DENV4 sequences from the NCBI protein database were studied for conservancy. In contrast to a high degree of conservancy within the DENV2 serotype, 35 out of the 42 epitopes did not occur in any of the 384 DENV-1, 3 and 4 sequences tested and only 7 epitopes had sequence homologues in one or more of the other serotypes. Interestingly, most of the epitopes which show conservancy across serotypes have been identified in the B*0702 transgenic mice. 4 of the identified B*0702 restricted epitopes (NS31682-1690, NS31700-1709, NS31753-1761, NS4B2280-2892) were additionally conserved in 89-100% of sequences derived from serotypes other than DENV2. The same has been observed for two DRB1*0101 restricted epitopes which were conserved across serotypes (NS31742-1756, NS52966-2980). Here, the epitopes were conserved in >99% of polyprotein sequences of two serotypes other than DENV2. Finally, one of the A*0101 restricted epitopes (NS11090-1099) is also conserved in 100% of DENV3 sequences. All results from this analysis are shown in Tables 2 and 3.
The DENV2 epitopes identified in Table 2 were analyzed for their respective homologues in DENV1, DENV3 and DENV4. 162 DENV1, 171 DENV2, 169 DENV3 and 53 DENV4 sequences from the NCBI Protein database were analyzed for conservancy. Table 3 shows the sequences of the epitopes identified after infection with DENV2 (bold letters). “Counts” indicate the number of strains in which the epitope is conserved within the respective serotype. Listed for each epitope are variants of the epitope in the DENV1, 3 and 4 serotypes and their respective counts. Epitopes are sorted according to their appearance in Table 2. These sequences help determine the cross-reactivity patterns of the identified epitopes.
E
451-459
ITEAELTGY
DENV2
146
NS1
1090-1099
RSCTLPPLRY
DENV2
171
NS2A
1192-1200
MTDDIGMGV
DENV2
143
NS2A
1251-1259
LTDALALGM
DENV2
156
NS4B
2399-2407
VIDLDPIPY
DENV2
90
NS5
3375-3383
YTDYMPSMK
DENV2
168
E
631-639
RLITVNPIV
DENV2
168
NS2B
1355-1363
IMAVGMVSI
DENV2
157
NS2B
1383-1391
GLLTVCYVL
DENV2
170
NS4A
2074-2083
RIYSDPLALK
DENV2
153
NS4B
2315-2323
ATVLMGLGK
DENV2
168
NS5
2608-2616
STYGWNLVR
DENV2
171
NS5
3079-3087
TVMDIISRR
DENV2
155
NS5
3112-3291
RQMEGEGVFK
DENV2
74
NS5
3283-3291
RTTWSIHAK
DENV2
111
NS2A
1212-1221
RPTFAAGLLL
DENV2
158
NS3
1682-1690
LPAIVREAI
DENV2
171
NS3
1700-1709
APTRVVAAEM
DENV2
170
NS3
1753-1761
VPNYNLIIM
DENV2
171
NS3
1808-1817
APIMDEEREI
DENV2
131
NS3
1978-1987
TPEGIIPSMF
DENV2
170
NS5
2966-2980
SRAIWYMWLGARFLE
DENV2
171
NS2B
1383-1391
LLVISGLFPV
DENV2
85
NS3
1465-1473
AAAWYLWEV
DENV2
157
NS3
1681-1689
YLPAIVREA
DENV2
170
NS3
2013-2022
DLMRRGDLPV
DENV2
157
NS4A
2140-2148
ALSELPETL
DENV2
169
NS4A
2205-2213
IILEFFLIV
DENV2
170
NS5
3058-3066
KLAEAIFKL
DENV2
162
NS3
1509-1517
SQIGAGVYK
DENV2
168
NS3
1608-1617
GTSGSPIIDK
DENV2
49
NS3
1863-1871
KTFDSEYVK
DENV2
129
NS3
2070-2078
KPRWLDARI
DENV2
155
NS4B
2280-2289
RPASAWTLYA
DENV2
171
NS4B
2296-2303
TPMLRHSI
DENV2
171
NS5
2646-2655
SPNPTVEAGR
DENV2
92
NS5
2885-2894
TPRMCTREEF
DENV2
152
NS5
3077-3085
RPTPRGTVM
DENV2
166
C
53-67
AFLRFLTIPPTAGIL
DENV2
169
NS2A
1199-1213
GVTYLALLAAFKVRP
DENV2
156
NS3
1356-1370
MAVGMVSILASSLLK
DENV2
171
NS3
1742-1756
TFTMRLLSPVRVPNY
DENV2
120
This example includes a discussion of the foregoing data and conclusions based upon the data.
Wild-type mice are resistant to DENV-induced disease, and therefore, development of mouse models for DENV infection to date has been challenging and has had to rely on infection of immunocompromised mice, non-physiologic routes of infection, and mouse-human chimeras (Yauch, et al. Antiviral Res 80:87 (2008)). Due to the importance of the IFN system in the host antiviral response, mice lacking the IFNR-α/β support a productive infection. A mouse-passaged DENV2 strain, S221, is highly immunogenic and also replicates to high levels in IFNR-α/β−/− mice, thus allowing the study of CD4+ and CD8+ T cell responses in DENV infection. In this murine model, vaccination with T cell epitopes prior to S221 infection provided significant protection (Yauch, et al. J Immunol 185:5405 (2010); Yauch, et al. J Immunol 182:4865 (2009)). While significant differences exist between human and murine TCR repertoires and processing pathways, HLA transgenic mice are fairly accurate models of human immune responses, especially when peptide immunizations are utilized. Numerous studies to date show that these mice develop T cell responses that mirror the HLA restricted responses observed in humans in context of various pathogens (Gianfrani, et al. Hum Immunol 61:438 (2000); Wentworth, et al. Eur J Immunol 26:97 (1996); Shirai, et al. J Immunol 154:2733 (1995); Ressing, et al. J Immunol 154:5934 (1995); Vitiello, et al. J Exp Med 173:1007 (1991); Diamond, et al. Blood 90:1751 (1997); Firat, et al. Eur J Immunol 29:3112 (1999); Le, et al. J Immunol 142:1366 (1989); Man, et al. Int Immunol 7:597 (1995)).
The data disclosed herein demonstrate that HLA transgenic IFNRα/βR−/− mice are a valuable model to identify DENV epitopes recognized in humans. Not only were a number of HLA-restricted T cell responses identified, but the genome wide screen provided further insight into the subproteins targeted by T cells during DENV infection. The majority of DENV responses (97%) were derived from the nonstructural proteins; more than half of the epitopes identified originate from the NS3 and NS5 protein. The data show the immunodominant role of the highly conserved NS3 protein (Rothman Adv Virus Res 60:397 (2003); Duangchinda, et al. Proc Natl Acad Sci USA 107:16922 (2010)), and also suggest NS5 as a major target of T cell responses. Interestingly, proteins previously described as antibody targets (prM, E and NS1) (Rothman J Clin Invest 113:946 (2004)) accounted for less than 5% of all responses, with only 3 epitopes identified from these proteins. The observation that T cell and B cell epitopes after primary DENV infection are not derived from the same proteins may factor in vaccine design, since immunizing with NS3 and NS5 T cell epitopes would induce a robust T cell response without the risk of antibody-dependent-enhancement (ADE).
Another unique challenge in vaccine development is the high degree of sequence variation in a pathogen, characteristically associated with RNA viruses. This is of particular relevance in the case of DENV infections, where it is well documented that prior exposure to a different serotype may lead to more severe disease and immunopathology (Sangkawibha, et al. Am J Epidemiol 120:653 (1984)). The fact that there is also significant genetic variation within each serotype adds to the complexity of successful vaccinations (Twiddy, et al. Virology 298:63 (2002); Holmes, et al. Trends Microbiol 8:74 (2000)). It is hypothesized that in certain cases, peptide variants derived from the original antigen in the primary infection, with substitutions at particular residues, can induce a response that is qualitatively different from the response induced by the original antigen (for example inducing a different pattern of lymphokine production; Partial agonism), or even actively suppressing the response (TCR antagonism). Variants associated with this phenotype are often collectively referred to as Altered Peptide Ligands (APLs) (Yachi, et al. Immunity 25:203 (2006)). During secondary infections, the T cell response directed at the APL may lead to altered or aberrant patterns of lymphokine production, and TCR antagonist mediated inhibition of T cell responses (Kast, et al. J Immunol 152:3904 (1994)). Therefore, immunity to all four serotypes would provide an optimal DENV vaccine. It is generally recognized that conserved protein sequences represent important functional domains (Valdar Proteins 48:227 (2002)), thus mutations at these important protein sites could be detrimental to the survival of the virus. T cell epitopes that target highly conserved regions of a protein are therefore likely to target the majority of genetic variants of a pathogen (Khan, et al. Cell Immunol 244:141 (2006)). Most interestingly in this context was that epitopes that are highly conserved within the DENV2 serotype are the major target for T cells. This data suggests, that immunizations with peptides from a given serotype would protect from the majority of genotypes within this serotype. In contrast, the DENV2 derived epitopes identified are not conserved in other serotypes. These findings point to an immunization strategy with a collection of multiple non-crossreactive epitopes derived from each of the major DENV serotypes. The induction of separate non-crossreactive responses would avoid issues arising from incomplete crossreaction and APL/TCR antagonism effects.
In addition to sequence variation, HLA polymorphism adds to the complexity of studying T cell responses to DENV. MHC molecules are extremely polymorphic, with several hundred different variants known in humans (Klein, Natural History of the Major Histocompatibility Complex (1986); Hughes, et al. Nature 355:402 (1992)). Therefore, selecting multiple peptides matching different MHC binding specificities will increase coverage of the patient population for diagnostic and vaccine applications alike. However, different MHC types are expressed at dramatically different frequencies in different ethnicities. To address this issue, IFNR-α/βR−/− mice were backcrossed with mice transgenic for HLA A*0101, A*0201, A*1101, B*0702 and DRB1*0101. These four MHC class I alleles were chosen as representatives of four supertypes (A1, A2, A3 and B7, respectively) and allow a combined coverage of approximately 90% of the worldwide human population (Sette, et al. Immunogenetics 50:201 (1999)), with more than 50% expressing the specific alleles. HLA supertypes are not limited to class I molecules. Several studies have demonstrated the existence of HLA class II supertypes (Doolan, et al. J Immunol 165:1123 (2000); Wilson, et al. J Virol 75:4195 (2001); Southwood, et al. J Immunol 160:3363 (1998)) and functional classification has revealed a surprising degree of repertoire sharing across supertypes (Greenbaum, et al. Immunogenetics 63:325 (2011)). This is in accordance with the data, since the DRB1*0101 restricted epitopes were identified in almost every donor, regardless if the donor was expressing the actual allele. Overall, the mouse model significantly reflects the response pattern observed in humans and that HLA B restricted responses seem to be dominant in B*0702 transgenic mice as well as in human donors, expressing the B*0702 allele (
The dominance of HLA B responses has been shown in context of several other viruses, such as HIV, EBV, CMV, and Influenza (Kiepiela, et al. Nature 432:769 (2004); Bihl, et al. J Immunol 176:4094 (2006); Boon, et al. J Immunol 172:4435 (2004); Lacey, et al. Hum Immunol 64:440 (2003)), suggesting that this observation is not limited to RNA viruses, and in fact, it has even been described for an intracellular bacterial pathogen, Mycobacterium Tuberculosis (Lewinsohn, et al. PLoS Pathog 3:1240 (2007); Axelsson-Robertson, et al. Immunology 129:496 (2010)). Furthermore, HLA B restricted T cell responses have been described to be of higher magnitude (Bihl, et al. J Immunol 176:4094 (2006)) and to influence infectious disease course and outcome. In case of DENV, one particular B07 epitope was reported to elicit higher responses in patients with DHF compared to patients suffering from DF only and could therefore be associated with disease outcome (Zivna, et al. J Immunol 168:5959 (2002)). Other reports suggest a role for HLA B44, B62, B76 and B77 alleles in protection against developing clinical disease after secondary DENV infection, whereas other alleles were associated with contribution to pathology (Stephens, et al. Tissue Antigens 60:309 (2002); Appanna, et al. PLoS One 5 (2010). Accordingly, HLA alleles appear to be associated with clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals. The fact that the stronger B*07 response occurs in our human samples as well as in our mouse model of DENV infection validates the relevance of this mouse model, since it even mimics patterns of immuno-dominance observed in humans.
This example includes a description of the identification of T cell responses against additional DENV-derived peptides in human donors.
Peripheral blood samples were obtained from healthy adult blood donors from the National Blood Center in Colombo, Sri Lanka. DENV-seropositivity was determined by ELISA. Those samples that are positive for DENV-specific IgM or IgG are further examined by the FACS based neutralization assay to determine whether the donor may have been exposed to single or multiple DENV serotypes. For MHC class I binding predictions all 9- and 10-mer peptides were predicted for their binding affinity to their respective alleles. Binding predictions were performed using the command-line version of the consensus prediction tool available on the IEDB web site. Peptides were selected if they were in the top 1% of binders.
As HLA typing and ELISA results were available, donor samples were tested such that predicted peptides for all four serotypes were tested against all appropriate and available HLA types expressed by the donor. DENV specific T cell responses were detected directly ex vivo from our Sri Lankan donor cohort, as measured by an IFNγELISPOT assay. All epitopes that have been identified in one or more donors are listed in Table 4.
This application is a U.S. National Phase of International Application No. PCT/US2012/044071, filed Jun. 25, 2012, which designated the U.S. and that International Application was published under PCT Article 21(2) in English, which is a continuation-in-part of application serial no. PCT/US2011/041889, filed Jun. 24, 2011, and claims priority to U.S. Provisional Application No. 61/391,882, filed Oct. 11, 2010 and U.S. Provisional Application No. 61/358,142, filed Jun. 24, 2010, all of which applications are incorporated herein by reference in their entirety.
This invention received government support from the National Institutes Health grants AI060989, AI077099, U54 AI057157, U01A082185 and National Institutes of Health Contract HHSN272200900042C. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/044071 | 6/25/2012 | WO | 00 | 4/7/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61391882 | Oct 2010 | US | |
| 61358142 | Jun 2010 | US |
