Patent Grant
10322171
This application includes as part of its disclosure a biological sequence listing text file named “49561o1204.txt” having a size of 54,202 bytes that was created Jul. 17, 2017, which is incorporated by reference in its entirety.
The invention generally relates to the development of a vaccine capable of providing protection against viral infection associated with antibody-dependent enhancement (ADE) of infection, such as Zika, Dengue and Ebola infections or other viruses where viral antigens may elicit ADE, as well as the production of antibodies to treat or prevent such infections. More particularly, the invention is related to the use of an information spectrum method (ISM) to identify novel peptides that possess sufficient structural homology with Zika envelope glycoprotein (GP1) to be suitable for use as an antigen to elicit an immune response, i.e., antibody production, but which elicit no or reduced activation of the complement system.
Zika is a disease caused by Zika virus, which is spread to people primarily through the bite of an infected Aedes species mosquito. The most common symptoms of Zika are fever, rash, joint pain, and conjunctivitis. The illness is usually mild (not severe enough to require hospitalization or result in death), with symptoms appearing 2 to 7 days after being bitten by an infected mosquito and lasting for several days to a week. However, there have been reports of serious birth defects, namely microcephaly, and other poor pregnancy outcomes in babies of mothers who were infected with Zika virus while pregnant. There have also been cases of Guillain-Barré syndrome (GBS) reported in patients following suspected Zika virus infection. GBS is a rare disorder where a person's own immune system damages the nerve cells, causing muscle weakness and sometimes, paralysis. These symptoms can last anywhere from a few weeks to several months, although some people have permanent damage and, in rare cases, GBS may result in death.
Zika virus is a member of the virus family Flaviviridae, and is thus related to dengue, yellow fever, Japanese encephalitis, and West Nile viruses. Like other members of the Flavivirus genus, Zika contains a positive single-stranded genomic RNA, encoding a polyprotein that is processed into three structural proteins (the capsid (C), the precursor of membrane (prM) and the envelope (E)) and seven nonstructural proteins (NS1 to NS5).
Zika virus is transmitted to people primarily through the bite of an infected Aedes species mosquito (A. aegypti and A. albopictus). These are the same mosquitoes that spread dengue and chikungunya viruses. These mosquitoes typically lay eggs in and near standing water in things like buckets, bowls, animal dishes, flower pots and vases. They prefer to bite people, and live indoors and outdoors near people. Mosquitoes that spread chikungunya, dengue, and Zika are aggressive daytime biters. They can also bite at night. Mosquitoes become infected when they feed on a person already infected with the virus. Infected mosquitoes can then spread the virus to other people through bites. Zika virus usually remains in the blood of an infected person for about a week but it can be found longer in some people.
Prior to 2015, Zika virus outbreaks occurred in areas of Africa, Southeast Asia, and the Pacific Islands. The first human Zika infection was reported in Uganda in 1964 and the virus was later isolated from humans in South East Asia. Despite this broad geographical distribution, human Zika infections remained sporadic and limited to small-scale epidemics for decades, until 2007, when a large epidemic was reported on Yap Island, a territory of the Federated States of Micronesia, with nearly 75% of the population being infected with the virus. Moreover, an outbreak of a syndrome due to Zika fever was reported in French Polynesia, in addition to several cases of Zika infection in New Caledonia, Easter Island and the Cook Islands, indicating a rapid spreading of the virus in the Pacific. Two imported cases of Zika infection of travellers from Indonesia and the Cook Islands, respectively, to Australia and two from Thailand to Europe and Canada, respectively, was described recently (see, Kwong et al. (2013) Am J Trop Med Hyg 89:516-517 Tappe et al. (2014) European communicable disease bulletin; Pyke et al. (2014) PLoS currents 6; Fonseca et al. (2014) Am J Trop Med Hyg 91:1035-1038), emphasizing the capacity of Zika to spread to non-endemic areas where the proper mosquito vector might be present.
In May 2015, the Pan American Health Organization (PAHO) issued an alert regarding the first confirmed Zika virus infections in Brazil. Currently, outbreaks are occurring in many countries. Zika virus will continue to spread and it will be difficult to determine how and where the virus will spread over time.
On Jan. 22, 2016, the Centers for Disease Control and Prevention (CDC) activated its Emergency Operations Center (EOC) to respond to outbreaks of Zika occurring in the Americas and increased reports of birth defects and Guillain-Barré syndrome in areas affected by Zika.
In Feb. 1, 2016, the World Health Organization declared a Public Health Emergency of International Concern (PHEIC) because of clusters of microcephaly and other neurological disorders in some areas affected by Zika.
On Feb. 8, 2016, CDC elevated its EOC activation to a Level 1, the highest level.
As of Feb. 17, 2016, in the United States, 84 cases of travel-associated Zika virus disease cases have been reported. With the recent outbreaks, the number of Zika cases among travelers visiting or returning to the United States will likely increase. These imported cases could result in local spread of tile virus in the United States.
There is currently no available vaccine to prevent or treat Zika infection. Accordingly, the identification of novel antigens that are capable of eliciting an immune response against Zika to provide protection against infection is desirable.
The invention provides a method for identifying novel antigens possessing structural homology with the envelope glycoprotein (GP) from a virus associated with antibody-dependent enhancement (ADE) of infection, such as Flaviviridae, Flavivirus, e.g., Dengue or Zika, and Filoviridae, e.g., an Ebolavirus, e.g., Zaire ebolavirus or Sudan ebolavirus, preferably Zika virus, which are capable of eliciting an immune response against the virus and which, moreover, possess structural properties that minimize or eliminate undesirable properties associated with GP antigens, i.e., the identified antigens induce no or minimal antigen-dependent enhancement (ADE) of infection as a result of eliciting no or fewer infectivity-enhancing antibodies that activate the complement system.
The invention also provides novel antigens identified by such methods, which antigens possess structural homology with an envelope glycoprotein (GP) from a virus associated With antibody-dependent enhancement (ADE) of infection, such as Flaviviridae. e.g., Flavivirus, Dengue or Zika, and Filoviridae, e.g., an Ebolavirus, e.g., Zaire ebolavirus or Sudan ebolavirus, preferably Zika virus and, thus, are capable of eliciting an immune response against the virus, but which preferably induce no or minimal antigen-dependent enhancement (ADE) of infection as a result of eliciting no or fewer infectivity-enhancing antibodies that activate the complement system. Such antigens can be used as the basis for a vaccine to treat or prevent virus infection, preferably Zika, Dengue or Ebola infection.
In one aspect, the invention provides a method of eliciting an immune response by administering an immunologic composition comprising one or more synthetic peptides having an informational spectrum (IS) containing a frequency component F(0.338), where the frequency component may include a range of +/−0.001, 0.002, 0.003, 0.004, or 0.005, e.g., F(0.333-0.343), the amplitude of which is attenuated as compared to the amplitude oldie frequency component F(0.338) contained in the IS of an envelope glycoprotein (GP) protein or fragment thereof of a virus associated with ADE, such as Zika, Dengue, and Ebola viruses, where the attenuation may include at least a 40% reduction in signal strength, e.g., a decrease in amplitude of the peak at F(0.338) by about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or a complete reduction (100%). The synthetic peptide or peptides are capable of eliciting an immune response against the GP protein or fragment thereof.
The GP protein or fragment and the synthetic peptide or peptides may be sufficiently homologous such that the synthetic peptide or peptides is/are suitable for use as an antigen to elicit the production of antibodies, preferably neutralizing antibodies, more preferably neutralizing antibodies without a C1q component, that bind to the GP protein or fragment thereof. In particular, the information spectrum (IS) of the synthetic peptide may contain a frequency component F(0.295), where the frequency component may include a range of +/−0.001, 0.002, 0.003, 0.004, or 0.005, F(0.290-0.300), i.e., a frequency that is conserved across virus. e.g., Zika, envelope glycoproteins proteins and encodes structural information that does not pertain to the interaction between the virus and the host complement system, i.e., C1q.
The synthetic peptide or peptides may have a reduced ability to elicit infectivity-enhancing antibodies, as compared to the ability of the GP protein or fragment thereof to elicit infectivity-enhancing antibodies. Alternatively, the synthetic peptide or peptides may not elicit infectivity-enhancing antibodies.
The synthetic peptide or peptides may induce no or minimal antigen-dependent enhancement (ADE) of infection.
The synthetic peptide or peptides may not interact with a complement protein, such as C1q, or have a reduced ability to interact with a complement protein, such as C1q. As a result, the synthetic peptide does not activate or has a reduced ability to activate the host complement system, which is involved in ADE.
In one aspect, the GP protein or fragment thereof comprises or consists of an amino acid sequence selected from:
In another aspect, the GP protein or fragment thereof comprises or consists of an amino acid sequence:
The synthetic peptide or peptides may comprise 150-300 amino acids. More particularly, the synthetic peptide or peptides comprise 250-285 amino acids. Even more particularly, the synthetic peptide or peptides comprise or consist of 282 amino acids.
The synthetic peptide or peptides may contain one or more amino acid substitutions relative to the amino acid sequence of the GP protein or fragment thereof. For example, the synthetic peptide or peptides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to the amino acid sequence of the GP protein or fragment thereof. The substitutions attenuate the amplitude of the frequency component F(0.338), which may include a range of +/−0.001, 0.002, 0.003, 0.004, or 0.005, e.g., F(0.333-0.343).
1. In one aspect, the amino acid substitution comprises replacing an amino acid residue in the GP protein or fragment thereof with an amino acid residue having a dissimilar EHP value. For example, the amino acid substitutions may include or consist of replacing an aspartic acid (D) with another amino acid residue, preferably a leucine (L) or an isoleucine (I). Alternatively, the amino acid substitutions may include or consist of replacing a leucine (L) or an isoleucine (I) with an aspartic acid (D). More particularly, the amino acid substitutions may include or consist of one or more of the following: (i) replacing the isoleucine (I) at position 21 of the GP protein with D, M, F, R; (ii) replacing the isoleucine (I) at position 37 of the GP protein with E, V, N, L, G; (iii) replacing the leucine (L) at position 43 of the GP protein with E, I, V, N; (iv) replacing the arginine (R) at position 75 of the GP protein with D, T, S, F; (v) replacing the leucine (L) at position 129 of the GP protein with E, N, G, I, V; (vi) replacing the leucine (L) at position 218 of the GP protein with E, V, I, G; (vii) replacing the leucine (L) at position 240 of the GP protein with D, F, S, R; (viii) replacing the isoleucine (I) at position 266 of the GP protein with E, G, N, L, N, V; and (ix) replacing the isoleucine (I) at position 269 of the GP protein with D, M, T, R. Moreover, the amino acid substitution may include or consist of replacing a leucine (L) with a proline (P), preferably L398P in Ebola GP1 (strain KM233035 in GenBank).
In another aspect, the synthetic peptide or peptides comprise or consist of an amino acid sequence selected from:
Additionally, the present invention encompasses an immunologic composition comprising a synthetic peptide or peptides having an informational spectrum (IS) containing a frequency component F(0.338), where the frequency component may include a range of +/−0.001, 0.002, 0.003, 0.004, or 0.005, e.g., F(0.333-0.343), the amplitude of which is attenuated as compared to the amplitude of the frequency component F(0.338) contained in the IS of an envelope glycoprotein (GP) protein or fragment thereof of a virus associated with ADE, e.g., Zika, Dengue and Ebola, preferably the Zika virus envelope glycoprotein (GP) protein or fragment thereof, where the attenuation may include at least a 40% reduction in signal strength, e.g., a decrease in amplitude of the peak at F(0.338) by about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or a complete reduction (100%), wherein the synthetic peptide or peptides are capable of eliciting an immune response against the GP protein or fragment thereof.
The synthetic peptide or peptides employed in the immunologic composition may have no ability or a reduced ability to elicit infectivity-enhancing antibodies, as compared to the ability of the GP protein or fragment thereof to elicit infectivity-enhancing antibodies; induce no or minimal antigen-dependent enhancement (ADE) of infection; have a reduced ability to interact with a complement protein, such as C1q; be sufficiently homologous to the Zika GP protein or fragment thereof that they are suitable for use as an antigen to elicit the production of antibodies (preferably neutralizing antibodies that, more preferably, also lack a C1q component); have an IS that contains a frequency component F(0.295), where the frequency component may include a range of +/−0.001, 0.002, 0.003, 0.004, or 0.005, e.g., F(0.290-0.300); comprise 150-300 amino acids or, more preferably, 250-285 amino acids or, even more preferably, comprise or consist of 282 amino acids; contain one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) relative to the amino acid sequence of the GP protein or fragment thereof (particular sequences of which are set forth above and provided herein), which substitutions attenuate the amplitude of the frequency component F(0.338), as set forth above. Zika virus envelope glycoprotein (GP) protein or fragment thereof, and (ii) produces antibodies that bind to a Zika virus GP protein or fragment thereof.
The synthetic peptide or peptides may have no ability or a reduced ability to elicit infectivity-enhancing antibodies, as compared to the ability of the GP protein or fragment thereof to elicit infectivity-enhancing antibodies; induce no or minimal antigen-dependent enhancement (ADE) of infection, have a reduced ability to interact with a complement protein, such as C1q; be sufficiently homologous to the viral GP protein or fragment thereof that they are suitable for use as an antigen to elicit the production of antibodies (preferably neutralizing antibodies that, more preferably, also lack a C1q component); have an IS that contains a frequency component F(0.295), where the frequency component may include a range of +/−0.001, 0.002, 0.003, 0.004, or 0.005, e.g., F(0.290-0.300); comprise 150-300 amino acids or, more preferably, 250-285 amino acids or, even more preferably, comprise or consist of 282 amino acids; contain one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) relative to the amino acid sequence of the GP protein or fragment thereof (particular sequences of which are set forth above and provided herein), which substitutions attenuate the amplitude of the frequency component F(0.338), as set forth above. The synthetic peptide or peptides may be suitable for use as an antigen in an immunologic composition, such as a vaccine, including a polyvalent vaccine.
The present invention further encompasses a method of using at least one synthetic peptide as an immunogen in order to generate antibodies, preferably neutralizing antibodies that, more preferably, lack a C1q component, that specifically bind to an envelope glycoprotein or fragment thereof of a virus associated with ADE, e.g., Zika, Dengue and Ebola, preferably the Zika virus envelope glycoprotein (GP) protein or fragment thereof.
Additionally, the present invention contemplates an isolated nucleic acid encoding the synthetic peptide; a vector containing the nucleic acid, and an isolated cell containing the vector as well as an antibody or antigen binding fragment thereof capable of binding to the synthetic peptide. Moreover, the invention further contemplates a therapeutic method or diagnostic method using the synthetic peptide or the antibody or antigen binding fragment thereof that binds to the synthetic peptide.
Furthermore, the present invention encompasses a method for identifying a peptide that elicits an immune response against an envelope glycoprotein or fragment thereof of a virus associated with ADE, e.g., Zika, Dengue and Ebola, preferably the Zika virus envelope glycoprotein (GP) protein or fragment thereof. The method comprises (1)(i) obtaining an amino acid sequence of an envelope glycoprotein or fragment thereof of a virus associated with ADE, e.g., Zika, Dengue and Ebola (ii) assigning an electron-ion interaction potential (EHP) index value to each amino acid residue contained in the amino acid sequence of the GP protein or fragment (iii) subjecting the resultant EHP index values to discrete Fourier transformation (DEA); (iv) generating an informational spectrum (IS) of the at least one GP protein or fragment based on the EHP index values; (2)(i) obtaining an amino acid sequence of a peptide or peptides; (ii) assigning an EHP index value to each amino acid residue contained in the amino acid sequence of the peptide or peptides; (iii) subjecting the resultant EHP index values to discrete Fourier transformation (DFT); (iv) generating an informational spectrum (IS) of each peptide based on the FHP index values; (3) comparing the IS of the GP protein or fragment generated in (1)(iv) to the IS of the peptide or peptides generated in (2)(iv) and (4) identifying the peptide or peptides whose IS contain a frequency component F(0.338), where the frequency component may include a range of +/−0.001, 0.002, 0.003, 0.004, or 0.005, e.g., F(0.333-0.343), the amplitude of which is attenuated as compared to the amplitude of the frequency component F(0.338) contained in the IS of the GP protein or fragment thereof, as one being capable of eliciting an immune response against the GP protein or fragment. In one aspect, the GP protein or fragment and the synthetic peptide are otherwise homologous.
The method may further include synthesizing at least one of the identified peptides; synthesizing at least one of the identified peptides assessing its immunogenicity or ability to generate antibodies, preferably neutralizing antibodies that, more preferably, lack a C1q component, that specifically bind to said at least one GP protein; and/or producing an immunologic composition comprising at least one of the identified peptides.
The synthetic peptide or peptides may have no ability or a reduced ability to elicit infectivity-enhancing antibodies, as compared to the ability of the GP protein or fragment thereof to elicit infectivity-enhancing antibodies; induce no or minimal antigen-dependent enhancement (ADE) of infection; have a reduced ability to interact with a complement protein, such as C1q; be sufficiently homologous to the Zika GP protein or fragment thereof that they are suitable for use as an antigen to elicit the production of antibodies (preferably neutralizing antibodies that, more preferably, also lack a C1q component); have an IS that contains a frequency component F(0.295), where the frequency component may include a range of +/−1-0.001, 0.002, 0.003, 0.004, or 0.005, e.g., F(0.290-0.300); comprise 150-300 amino acids or, more preferably, 250-285 amino acids or, even more preferably, comprise or consist of 282 amino acids; contain one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) relative to the amino acid sequence of the GP protein or fragment thereof (particular sequences of which are set forth above and provided herein), which substitutions attenuate the amplitude of the frequency component F(0.338), as set forth above. The synthetic peptide or peptides may be suitable for use as an antigen in an immunologic composition, such as a vaccine, including a polyvalent vaccine.
The present invention also contemplates a method for the treatment or prevention of Zika virus infection in a subject, comprising administering a therapeutically effective amount of the immunologic composition or a therapeutically effective amount of the synthetic peptide or a therapeutically effective amount of the antibody or antigen binding fragment thereof to the subject in need thereof preferably the treated subject is a human, such that the immunologic composition or the synthetic peptide or the antibody or antigen binding fragment thereof treat or prevent Zika infection in the subject.
The present invention provides a novel approach to the identification of novel peptides whose EHP structure, but not necessarily sequence, mimic a viral antigenic target (e.g., the envelope glycoprotein) of virus associated with ADE, e.g., Zika, Dengue and Ebola, preferably the Zika virus envelope glycoprotein (GP) protein or fragment thereof, and, thus, can be used to elicit an immune response (e.g., antibody production) against Zika virus. However, the identified antigenic peptides have an attenuated interaction with C1q and/or a reduced ability to activate the complement system, as compared to a viral antigenic target (e.g., the envelope glycoprotein) and, thus, avoid or minimize the adverse effects of a vaccine based thereon (e.g., the identified antigenic peptides do not induce or induce only minimal enhancement of infection).
The EHP structure refers to the electron-ion interaction potential and is unrelated to the linear amino acid sequence (primary structure) and peptide folding motifs (secondary and tertiary structure). In particular, the peptides share EHP homology with at least one viral envelope glycoprotein (GP) protein or fragment thereof and, thus, can be used as an antigen (e.g., in a prophylactic or therapeutic vaccine). As a result of the EHP structural similarity between the identified synthetic peptide and the natural protein, antibodies provided against the synthetic peptide are also capable of recognizing the naturally-occurring viral, e.g., Zika, GP protein. Accordingly, synthetic peptides whose structure mimics that of the viral, e.g., Zika, GP protein can be used to elicit an immune response against infection with the virus while minimizing the undesirable properties associated with tile viral, e.g., Zika, GP protein itself, i.e., the GP protein is characterized, at least in part, by a frequency component of F(0.338) which is responsible for interaction between the virus and the complement system and, thus, may play a role in eliciting infectivity-enhancing antibodies, which are not desirable.
The EHP structure that is used as the basis for peptide selection for this invention relies on the substitution of amino acids occurring within GP1 with amino acids having dissimilar EHP values. The following chart lists the EHP values for the amino acids:
Based on the EHP values, the following groupings are used to designate EHP structurally similar peptides. The group L, I, N, G, V, and E (EHP<0.0058) may be interchangeable with one another, and more particularly, the subgroup L, I, N, G, and V (EHP<0.0057) and the subgroup G and E (0.0050≤EHP≤0.0058). The group P, H, K, and A (0.0198≤EHP≤0.0373) may be interchangeable with one another, and more particularly the subgroup P, H, and K (0.0198≤EHP≤0.0371), the subgroup P and H (0.0198≤EHP≤0242), and the subgroup K and A (0.371≤EHP≤0.373). The group Y and W may be interchangeable (0.0516≤EHP≤0.0548). The group Q, M, S, and C (0.0761≤EHP≤0.0829) may be interchangeable. The group T, F, and R (0.0941≤EHP≤0.0959) may be interchangeable. In general, D (EHP=0.1263) is not interchangeable with other amino acids.
The amino acid substitutions employed in the subject invention are “incompatible” in that the substitutions are made across these groups, i.e., not “interchangeable” as set forth herein. For example, “dissimilar” amino acid substitutions may include or consist of replacing D with another amino acid residue, preferably L or I, replacing L or I with D; replacing I with D, M, T, F, or R; replacing R with D, T, S, or F; replacing I, with D, F, S, or R. By substituting an amino acid at a particular position within the naturally-occurring Zika GP protein with a dissimilar amino acid residue, the ability of the Zika GP protein mimic to interact with the complement system C1q) and elicit infectivity-enhancing antibodies is ablated, in whole or in part. The ability of the amino acid substitution(s) to remove or reduce this undesirable property is reflected in a reduction (attenuation) in the amplitude of the frequency component F(0.338).
In particular, the inventors have shown that substituting at least 3, at least 5 or at least 9 amino acid residues within the Zika virus GP1 Zika isolate AMA 12087 obtained in Brazil during 2015) with one or more dissimilar amino acid residues can attenuate the amplitude of the frequency component F(0.338) of the resultant peptide, thereby demonstrating that the “modified” peptide (which maintains sufficient homology with the Zika GP1 such that the synthetic peptide is suitable for use as an antigen to elicit the production of antibodies that bind to the GP protein present in the host following Zika infection) may have reduced interaction with the complement system (such as C1q) and, thus, is less capable of eliciting infectivity-enhancing antibodies, as compared to the ability of the GP protein or fragment thereof to elicit infectivity-enhancing antibodies, or, preferably, does not elicit infectivity-enhancing antibodies and, thereby, induces no or minimal antigen-dependent enhancement (ADE) of infection.
Zika is an emerging global health concern, with 32 countries and territories across various regions currently experiencing active Zika virus transmission. Given its disease-causing potential, particularly birth defects caused thereby, the prevention of Zika infection is a high public health priority. Currently, the most effective single way of protecting people against Zika infection and disease is to avoid traveling to countries where Zika virus is found and/or to avoid mosquito bites (e.g., wearing protective clothing, using insecticides, and/or eliminating mosquito breeding sites).
Zika is a little known emerging mosquito-borne Flavivirus, belonging to the Flaviviridae family that is closely related to the Spondweni serocomplex. Like other members of the Flavivirus genus, Zika virus contains a positive single-stranded genomic RNA that encodes, among other things, a structural envelope protein. The envelope glycoprotein (GP), embedded in the membrane and organized as a dimer, allows attachment of the virus particle to the host cell receptor to initiate infection.
Antibodies against the envelope glycoprotein (GP) are likely important for protection against Zika infection. For example, there is a safe and of vaccine against another flavivirus, yellow fever virus, based on the yellow fever F glycoprotein. However, the Zika virus GP has properties that suggest some antibodies specific to this target may enhance viral infectivity. In particular, the envelope glycoprotein from Ebola (and Dengue) are characterized by an informational spectrum (IS) containing a frequency component F(0.338). It has been shown that F(0.338) encodes structural information of the Ebola virus envelope glycoprotein that is responsible for interaction between the virus and the host complement system, i.e., C1q, thereby eliciting antigen-dependent enhancement (ADE) of infection. The inventors showed that introduction of a point mutation (L398R) into the Ebola GP1 attenuated the amplitude of F(0.338). Surprisingly, the inventors discovered that the Zika virus GP is also characterized by an IS containing F(0.338), suggesting that antibodies raised against this antigen may enhance viral infectivity.
In part, based on this observation, the present invention uses an ISM approach, rather than traditional neutralization assays, to identify structural characteristics of Ebola, Dengue, and Zika peptides (e.g., GP1) and, based thereon, obtain novel peptide antigens having a spectral profile overlapping, in part, with the spectral profile of the Ebola, Dengue, and Zika peptide(s), but also differing in that the desired novel peptide antigens have an IS that contains a frequency component F(0.338), the amplitude of which is attenuated (reduced) as compared to the amplitude of the frequency component F(0.338) contained in the IS of the envelope glycoprotein (GP) protein or fragment thereof encoded by an Ebola, Dengue, and Zika virus isolate.
Using this information, the inventors have identified synthetic peptides that differ in amino acid sequence from the naturally-occurring viral. e.g., Zika, peptides but possess the same or similar structural features as the viral protein(s) that are important for interacting with the host cell. As such, these synthetic peptides can be used as a novel antigen(s), e.g., capable of eliciting an immune response against the viral, e.g., Zika, protein or fragment thereof, e.g., suitable for use as an antigen to produce antibodies (preferably, neutralizing antibodies) that bind to the at least one Zika GP protein or fragment thereof. Additionally, because the synthetic peptides contain an IS in which the frequency component F(0.338) has an attenuated (reduced) amplitude as compared to the amplitude of the frequency component F(0.338) contained in the IS of the GP protein or fragment thereof encoded by a Zika vials isolate, the synthetic peptides effectively possess a reduced likelihood of eliciting infectivity-enhancing antibodies as compared to the ability of the GP protein or fragment thereof to elicit infectivity-enhancing antibodies, preferably the synthetic peptides do not elicit infectivity-enhancing antibodies, and/or induce no or minimal antigen-dependent enhancement (ADE) of infection and/or do not interact with a complement protein or have a reduced ability to interact with a complement protein (such as C1q), while retaining the ability to elicit the production of antibodies that bind to the GP protein or fragment thereof. In other words, the identified antigenic peptides mimic the naturally-occurring Zika peptide but without the undesirable properties of the natural peptide.
In the ISM approach, sequences (protein or nucleotide) are transformed into signals by assigning a numerical value to each element (amino acid or nucleotide). These values correspond to the electron-ion interaction potential (EHP), which determines electronic properties of amino acids and nucleotides. The signal obtained is then decomposed into a periodical function by Fourier transformation, resulting in a series of frequencies (F) and their amplitudes (A). The obtained frequencies correspond to the distribution of structural motifs with defined physico-chemical characteristics that are responsible for the biological function of the sequence. In other words, the peak frequencies of IS of a protein sequence reflect its biological or biochemical functions. See Veljkovic et al., Current Medicinal Chemistry (2007) 14:133-135, which is herein incorporated by reference in its entirety.
When comparing proteins that share the same biological or biochemical function(s), this technique allows the detection of code/frequency pairs that are specific for their common biological properties. The method is insensitive to the location of the motifs and, thus, does not require previous alignment of the sequences.
More particularly, it is generally believed that the number of valence electrons and the EHP representing the main energy term of valence electrons are essential physical parameters determining the long-range properties of biological molecules. EHP for organic molecules can be determined by the following simple equation derived from the “general model pseudopotential”:
W=0.25Z*sin(1.04pZ*)/2π
where Z* is the average quasi-valence number (AQVN) determined by
Z*=ΣmniZ1/N
wherein Zi is the valence number of the i-th atomic component, ni is the number of atoms of the i-th component, m is the number of atomic components in the molecule, and N is the total number of atoms. The EHP values calculated according to equations (1) and (2) are in Rydbergs (Ry). The strong connection between EHP and AQVN of organic molecules and their biological activity has previously demonstrated, e.g., in the context of mutagenicity, carcinogenicity, toxicity, antibiotic activity, and cytostatic activity.
A sequence of N residues is represented as a linear array of N terms, with each term given a weight. The weight assigned to a residue is EHP, determining electronic properties of amino acids and nucleotides, which are responsible for their intermolecular interactions. In this way the alphabetic code of protein or nucleotide sequence is transformed into a sequence of numbers. The obtained numerical sequence, representing the primary structure of protein, is then subjected to a discrete Fourier transformation, which is defined as follows:
X(n)=Σx(m)e−j(2/N)mn, n is 1, 2, . . . , N/2
Where x(m) is the m-th member of a given numerical series, N is the total number of points in this series, and X(n) are discrete Fourier transformation coefficients. These coefficients describe the amplitude, phase and frequency of sinusoids, which comprise the original signal. The absolute value of complex discrete Fourier transformation defines the amplitude spectrum and the phase spectrum. The complete information about the original sequence is contained in both spectral functions.
In this way, sequences are analyzed as discrete signals. It is assumed that their points are equidistant with the distance d is 1. The maximal frequency in a spectrum defined in this way is F is ½d is 0.5. The frequency range is independent of the total number of points in the sequence. The total number of points in a sequence influences only the resolution of the spectrum. The resolution of the N-point sequence is 1/n. The n-th point in the spectral function corresponds to a frequency f(n) is of is n/N. Thus, the initial information defined by the sequence of amino acids can now be presented in the form of the informational spectrum (IS), representing the series of frequencies and their amplitudes.
The IS frequencies correspond to distribution of structural motifs with defined physicochemical properties determining a biological function of a protein. When comparing proteins, which share the same biological or biochemical function, the ISM technique allows detection of code/frequency pairs which are specific for their common biological properties, or which correlate with their specific interaction. This common informational characteristic of sequences is determined by a cross-spectrum (CS). A CS of N spectra is obtained by the following equation:
C(j)πS(i,j)
where π (i,j) is the j-th element of the i-th power spectrum and C(j) is the j-th element of CS.
Thus, CS is the Fourier transform of the correlation function for the spectrum. In this way, any spectral component (frequency) not present in all compared informational spectra is eliminated. Peak frequencies in CS are common frequency components for the analyzed sequences. A measure of similarity for each peak is the signal-to-noise ratio (S/N), which represents a ratio between signal intensity at one particular IS frequency and the main value of the whole spectrum. If one calculates the CS for a group of proteins, which have different primary structures, and finds strictly defined peak frequencies, it means that primary structures of the analyzed proteins encode the same information. It was demonstrated that: 1) such a peak exists only for the group of proteins with the same biological function; 2) no significant peak exists for biologically unrelated proteins; 3) peak frequencies are different for different biological functions. Furthermore, it was shown that the proteins and their targets (ligand/receptor, antibody/antigen, etc.) have the same characteristic frequency(ies) in common. Thus, it can be postulated that IS frequencies characterize not only the general function but also recognition and interaction between a particular protein and its target. Once the characteristic frequency for a particular protein function/interaction is identified, it is possible then to utilize the ISM approach to predict the amino acids in the sequence, which essentially contribute to this frequency and are likely to be crucial for the observed function.
The calculation of the IS and CS of the amino acid sequence from Zika virus GP allowed the identification of conserved domains, e.g., structural properties, that likely play a role in the interaction of the viral GP protein with the cellular receptor.
The entry of Zika virus into susceptible cells is mediated, at least in part, by the viral envelope glycoprotein. In the present invention. ISM was applied to identify the spectral properties of GP proteins from Zika virus isolates and, further, to identify undesirable spectral properties of the GP proteins, namely a high amplitude frequency component F(0.338), which is associated with facilitating the interaction between Zika and the complement system, C1q. The ISM was successfully applied in structure-function analysis of different protein sequences, but may also be used in de nova design of peptides.
Using this information, the inventors have identified synthetic peptides that differ in amino acid sequence from the naturally-occurring Zika peptides such that the synthetic peptides lack undesirable traits associated with the naturally-occurring Zika peptides, i.e., induction of ADE, but possess the same or similar structural features as the viral protein(s) that are important for interacting with the host cell. As such, these synthetic peptides can be used as a novel antigen(s), e.g., capable of eliciting an immune response against the Zika GP protein or fragment thereof, e.g., suitable for use as an antigen to produce antibodies (preferably, neutralizing antibodies) that bind to the at least one Zika GP protein or fragment thereof.
The synthetic peptides of the present invention can be administered by different methods, e.g., topically, intranasally, or through parenteral administration, such as through subcutaneous injection, intra-muscular injection, intravenous injection, intraperitoneal injection, or intradermal injection, to a subject in need thereof, e.g., humans, horses, swine, canine and other mammals or avians, etc. The peptides can be used individually or in combination. Additionally, the peptide may be administered alone or as part of a composition that further comprises one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the peptide, chosen route of administration and standard biological administration. Because inventive peptides may target proteins on the surfaces of the virus and/or the cell, to ensure efficacy, the carrier in such formulations optionally are free or substantially free at least 90, 95, 98, or 99 wt %) of proteins that bind to the peptides.
Suitable pharmaceutically acceptable carriers for the compositions containing the peptides are described in the standard pharmaceutical texts. See, e.g., “Remington's Pharmaceutical Sciences”, 18th Ed., Mack Publishing Company, Easton, Pa. (1990). Specific non-limiting examples of suitable pharmaceutically acceptable carriers include water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance tile antiviral effectiveness of the composition.
Depending on the route of administration, the composition may take the form of a solution, suspension, tablet, pill, capsule, sustained release formulation, powder, cream, lotion, emulsion, or the like.
For topical administration, the antiviral peptide can be formulated into a composition containing an effective amount of the antiviral peptide, typically 0.01 or 0.1 to 10%, of the peptide. Such compositions are typically in the form of a solution, cream, lotion, or emulsion.
For parenteral administration, the peptides of tile present disclosure may be administered by intravenous, subcutaneous, intramuscular, intraperitoneal, or intradermal injection, alone or in compositions further comprising pharmaceutically accepted carriers. For administration by injection, it is preferred to use the antiviral peptide in a solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic. The antiviral peptides of the present disclosure can be obtained in the form of therapeutically acceptable salts that are well-known in the art.
The optimal concentration of the peptide or peptides will necessarily depend upon the specific peptide(s) used, the characteristics oldie patient, and the nature or particular serotype of the virus. These factors can be determined by those of skill in the medical and pharmaceutical arts in view of the present disclosure. In general, the peptides are most desirably administered at a concentration level that will generally afford antiviral effective results against the selected virus(es) without causing any harmful or deleterious side effects. Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drugs. Any of the above dosage forms containing effective amounts are well within the bounds of routine experimentation and therefore, well within the scope of the instant disclosure. A therapeutically effective dose may vary depending upon the route of administration and dosage form.
It will be further appreciated that the amount of an antiviral peptide of the present disclosure that is useful will vary not only with the particular peptide selected but also with the route of administration, and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or veterinarian. In general however, a suitable dose will be in the range of from about 0.01 to 750 mg/kg of body weight per day preferably in the range of 0.1 to 100 mg/kg/day, most preferably in the range of 0.5 to 25 mg/kg/day.
The peptides of the present disclosure may be administered therapeutically or prophylactically. Treatment is preferably commenced before or at the time of infection or at the time the mammal is exposed to a virus that is capable of causing infection, and continued until virus is no longer present or active. However, the treatment can also be commenced post-infection, after the mammal has been exposed to a virus that is capable of causing infection, or after the appearance of established symptoms of infection.
Suitable treatment is given 1-4 times daily and continued for 1-10 days, and typically 8 days post infection. Suitable prophylactic administration is given in single or multiple doses that may be spaced apart in accordance with known booster vaccination schedules and/or given annually.
The desired dose may be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day.
The peptide may be conveniently administered in unit dosage form, for example, containing 10 to 1500 mg, conveniently 20 to 1000 mg, most conveniently 50 to 700 mg of active ingredient per unit dosage form, e.g. 1 mg/kg equates to 75 mg/75 kg of body weight.
The present peptides can also be provided as pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” includes a salt with an inorganic base, organic base, inorganic acid, organic acid, basic amino acid, or acidic amino acid. As salts of inorganic bases, the disclosure includes, for example, alkali metals such as sodium or potassium, alkaline earth metals such as calcium and magnesium or aluminum; and ammonia. As salts of organic bases, the disclosure includes, for example, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, and triethanolamine. As salts of inorganic acids, the instant disclosure includes, for example, hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid. As salts of organic acids, the instant disclosure includes, for example, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. As salts of basic amino acids, the instant disclosure includes, for example, arginine, lysine and ornithine. Acidic amino acids include, for example, aspartic acid and glutamic acid.
The following examples are offered to illustrate, but not to limit, the claimed invention.
ISM was performed as described above. Generally, the analysis comprised the following steps:
1. each amino acid sequence was converted to the numerical sequence by representing each amino acid with the corresponding value of the EHP;
2. this numerical sequence was converted into a numerical spectrum using fast Fourier transform (FFT), and
3. spectra were mutually compared using cross-spectral analysis with the aim to extract common frequency components.
From the ISM analysis of human C1q(c) and GP1 from the different viruses (see
ISM was performed as described above. Generally, the analysis comprised the following steps:
1. each amino acid sequence was converted to the numerical sequence by representing each amino acid with the corresponding value of the EHP;
2. this numerical sequence was converted into a numerical spectrum using fast Fourier transform (FFT), and
3. spectra were mutually compared using cross-spectral analysis with the aim to extract common frequency components.
From the ISM analysis of human C1q(c) and GP1 from the different viruses (see
ISM was performed as described above. Generally, the analysis comprised the following steps:
1. each amino acid sequence was converted to the numerical sequence by representing each amino acid with the corresponding value of the EHP;
2. this numerical sequence was converted into a numerical spectrum using fast Fourier transform (FFT), and
3. spectra were mutually compared using cross-spectral analysis with the aim to extract common frequency components.
From the ISM analysis of Zika virus GP1 modified to contain different point mutations (see
ISM was performed as described in Veljokvic et al. (Feb. 19, 2015) In silico analysis suggests interaction between Ebola virus and the extracellular matrix. Frontiers in Microbiology 6(135):1-11, which is herein incorporated by reference in its entirety. Generally, the analysis comprised the following steps:
1. each amino acid sequence was converted to the numerical sequence by representing each amino acid with the corresponding value of the EHP;
2. this numerical sequence was converted into a numerical spectrum using fast Fourier transform (FFT); and
3. spectra were mutually compared using cross-spectral analysis with the aim to extract common frequency components.
From the ISM analysis of Ebola virus GP1 and elastin microfibrillar interface proteins (EMILINs), the CS of C1q and GP1 of EBOV strains KM233035, representing 99 of 101 GP1 homologous sequences from the outbreak 2014 in GenBank, as well as KJ660346 and KJ660348. This CS is characterized by a single peak corresponding to the IS frequency F(0.338), which represents the common information encoded by primary structures of GP1 and C1q (See
The mapping analysis was performed as described in Veljokvic et al. (Feb. 19, 2015) In silico analysis suggests interaction between Ebola virus and the extracellular matrix. Frontiers in Microbiology 6(135):1-11, which is herein incorporated by reference in its entirety.
Briefly, to identify the domain which is essential for information corresponding to the IS frequency F(0.338), the computer scanning of the primary structure of Ebola GP1 (KM233035) with peptides of different lengths was performed. This analysis showed that the main contribution to the frequency F(0.3138) comes primarily from the domain 341-375 (a.a. numbering in maturated protein without residues of the signal peptide) (
This application claims priority to and benefit of U.S. Provisional Application No. 62/297,713 filed Feb. 19, 2016, the disclosures of which are herein incorporated by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5705188 | Junichi et al. | Jan 1998 | A |
| 9267947 | Torres et al. | Feb 2016 | B2 |
| Number | Date | Country |
|---|---|---|
| 9730731 | Aug 1997 | WO |
| Entry |
|---|
| Dong and Liang, Virologica Sinica, 2018, 33:125-130. (Year: 2018). |
| Paessler and Veljkovic, F1000Research 2018, 7:298, 11 pages. (Year: 2018). |
| Pahil et al., PLoS One, 2017, 12(8):e0180505, 26 pages. (Year: 2018). |
| Amemiya K, et al. “Interleukin-12 induces a Th1-like response to Burkholderia mallei and limited protection in BALB/c mice,” Vaccine. Feb. 27, 2006;24(9)1413-20. |
| Amemiya K, et al. “Nonviable Burkholderia mallei induces a mixed Th1- and Th2-like cytokine response in BALB/c mice,” Infect Immun. May 2002;70(5):2319-25. |
| Bandara AB, et al. “A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice,” Microb Pathog. Sep. 2008;45(3):207-16. |
| Bondi SK, et al. “Strategies toward vaccines against Burkholderia mallei and Burkholderia pseudomallei,” Expert Rev Vaccines. Nov. 2008;7(9):1357-65. |
| Brett PJ, et al. “Structural and immunological characterization of Burkholderia pseudomallei O-polysaccharide-flagellin protein conjugates,” Infect Immun. Jul. 1996;64(7):2824-8. |
| Brett PJ, et al. “Isolation and characterization of Pseudomonas pseudomallei flagellin proteins,” Infect Immun. May 1994;62(5):1914-9. |
| Bryan LE, et al. “Passive protection of diabetic rats with antisera specific for the polysaccharide portion of the lipopolysaccharide isolated from Pseudomonas pseudomallei,” Can J Infect Dis. Jul. 1994;5(4):170-8. |
| Burtnick MN, et al. “The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei,” Infect Immun. Apr. 2011;79(4)1512-25. |
| Burtnick MN, et al. “Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages,” Infect Immun. Jan. 2010;78(1):88-99. |
| Choh LC, et al. “Burkholderia vaccines: are we moving forward?” Front Cell Infect Microbiol. Feb. 5, 2013;3:5. |
| Dance DA. “Melioidosis: the tip of the iceberg?” Clin Microbiol Rev. Jan. 1991;4(1):52-60. |
| Duval CW, et al. “The Histological Lesions of Experimental Glanders,” J Exp Med. Jul. 17, 1907;9(4):352-80. |
| Galyov EE, et al. “Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis,” Annu Rev Microbiol. 2010;64:495-517. |
| Horn JK. “Bacterial agents used for bioterrorism,” Surg Infect (Larchmt). 2003 Fall;4(3):281-7. |
| Hornstra H, et al. “Molecular epidemiology of glanders, Pakistan,” Emerg Infect Dis. Dec. 2009;15(12):2036-9. |
| Howe C, et al. “Human glanders; report of six cases,” Ann Intern Med. Jan. 1947;26(1):93-115. |
| Judy BM, et al. “Prophylactic application of CpG oligonucleotides augments the early host response and confers protection in acute melioidosis,” PLoS One. 2012;7(3):e34176. |
| Kespichayawattana W, et al. “Burkholderia pseudomallei induces cell fusion and actin-associated membrane protrusion: a possible mechanism for cell-to-cell spreading,” Infect Immun. Sep. 2000;68(9):5377-84. |
| Khan et al. “Glanders in animals: a review on epidemiology, clinical presentation, diagnosis and countermeasures.” Transboundary and emerging diseases. Jun. 2013;60(3):204-21. |
| Malik et al. “Emergence and re-emergence of glanders in India: a description of outbreaks from 2006 to 2011” Vet Ital. Apr. 1, 2012;48(2):167-78. |
| Massey et al. “In vivo bioluminescence imaging of Burkholderia mallei respiratory infection and treatment in the mouse model.” Frontiers in microbiology. Aug. 26, 2011;2:174. |
| Mott et al. “Characterization of the Burkholderia mallei tonB mutant and its potential as a backbone strain for vaccine development.” PLoS neglected tropical diseases. Jun. 26, 2015;9(6):e0003863. |
| Mougous et al. “A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus.” Science. Jun. 9, 2006;312(5779):1526-30. |
| Nelson et al. “Evaluation of lipopolysaccharide and capsular polysaccharide as subunit vaccines against experimental melioidosis.” Journal of medical microbiology. Dec. 1, 2004;53(12):1177-82. |
| Neubauer et al. “Serodiagnosis of Burkholderia mallei Infections in Horses: State-of-the-art and Perspectives.” Journal of Veterinary Medicine, Series B. Jun. 2005;52(5):201-5. |
| Nieves et al. “A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection.” Vaccine 29, 8381-8389 (2011). |
| Pukatzki et al. “Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system.” Proceedings of the National Academy of Sciences. Jan. 31, 2006;103(5):1528-33. |
| Sarkar-Tyson et al. “Progress toward development of vaccines against melioidosis: a review.” Clinical therapeutics. Aug. 1, 2010;32(8):1437-45. |
| Schell et al. “Type VI secretion is a major virulence determinant in Burkholderia mallei.” Molecular microbiology. Jun. 2007;64(6):1466-85. |
| Scholz et al. “Genotyping of Burkholderia mallei from an outbreak of glanders in Bahrain suggests multiple introduction events.” PLoS neglected tropical diseases. Sep. 25, 2014;8(9):e3195. |
| Silva et al. “Development of Burkholderia mallei and pseudomallei vaccines.” Frontiers in cellular and infection microbiology. Mar. 11, 2013;3:10. |
| Silva et al. “Correlates of immune protection following cutaneous immunization with an attenuated Burkholderia pseudomallei vaccine.” Infection and immunity. Oct. 7, 2013:IAI-00915. |
| Srinivasan et al. “Glanders in a military research microbiologist.” New England Journal of Medicine. Jul. 26, 2001;345(4):256-8. |
| Stone R. “Racing to defuse a bacterial time bomb.” Science. Aug. 24, 2007;317(5841):1022-4. |
| Turner et al. “Construction and characterization of genetically definedaro omp mutants of Enterotoxigenic Escherichia coli and preliminary studies of safety and immunogenicity in humans.” Infection and immunity. Aug. 1, 2001;69(8):4969-79. |
| Ulrich et al. “Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice.” Vaccine. Mar. 14, 2005;23(16):1986-92. |
| Van Zandt KE. Glanders: an overview of infection in humans. Orphanet journal of rare diseases. Dec. 2013;8(1):131. |
| Verma, A.K. et al. Glanders: A re-emerging zoonotic disease: A review. Journal of Biological Sciences 14.1 (2014): 38-51. |
| Vollmer J, et al. “Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities,” Eur J Immunol. Jan. 2004;34(1):251-62. |
| Wheelis M. “First shots fired in biological warfare,” Nature. Sep. 17, 1998;395(6699):213. |
| Whitlock GC, et al. “Glanders: off to the races with Burkholderia mallei,” FEMS Microbiol Lett. Dec. 2007;277(2):115-22. |
| Whitlock GC, et al. “Construction of a reporter system to study Burkholderia mallei type III secretion and identification of the BopA effector protein function in intracellular survival,” Trans R Soc Trop Med Hyg. Dec. 2008;102 Suppl 1: S127-33. |
| Whitlock GC, et al. “Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge,” Procedia Vaccinol. 2010;2(1). |
| Number | Date | Country | |
|---|---|---|---|
| 20170312354 A1 | Nov 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62297713 | Feb 2016 | US |
