Patent Application
20180021425
Herpesviridae is a large family of DNA viruses that cause diseases in animals, including humans. The members of this family are also known as herpesviruses.
All herpesviruses are composed of relatively large double-stranded, linear DNA genomes encoding 100-200 genes encased within an icosahedral protein cage called the capsid which is itself wrapped in a protein layer called the tegument containing both viral proteins and viral mRNAs and a lipid bilayer membrane called the envelope. This whole particle is known as a virion.
Cytomegalovirus (CMV)-based vaccines, as well as other herpesvirus-based vaccines, are on the horizon as a promising addition to our arsenal against infectious disease and cancer. These herpesvirus-based vectors are unique, not only in the high level of T cell immunity they induce against their heterologous encoded pathogen (or cancer) target antigen, but also in the durability of the immunity and in its ‘immediate-effector’ quality.
CMV is a member of the beta subclass of the herpesvirus family. To date, CMV is the best characterized of the herpesvirus-based vaccine vectors.
A RhCMV-based vaccine against the monkey version of HIV (simian immunodeficiency virus, SIV) was recently shown to induce protection against systemic infection in rhesus macaques—a level of protection never observed before for any SIV vaccination regimen.
CMV-based vaccines have also recently been shown to protect against such diverse pathogens and diseases as Ebola virus, tetanus and prostate cancer in mouse model systems using mouse CMV (MCMV).
Although CMV-based (and other less developed herpesvirus-based) vaccines have been shown to induce substantial levels of T cell responses against the encoded heterologous target antigens, the induction of substantial antibody responses against these targets has not been achieved by these vectors. This T cell bias is regarded as a limitation of these vaccines for their broad application to target infectious diseases and cancer. A number of references (patents and publications) are supplied as evidence supporting the generalized inability of herpesvirus-based vaccines to induce antibody responses.
The present invention relates generally to immune responses and more specifically to promoter usage providing differential immune response modulation.
All documents cited or referenced herein and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
The present invention is derived from unexpected findings from our Ebola virus challenge study in rhesus macaques vaccinated with a rhesus CMV expressing an Ebola virus target antigen (Ebola virus glycoprotein, GP).
A characteristic of herpesvirus genes is that they differ in their kinetics of expression in relationship to the herpesvirus replication cycle. Based on the time of expression, herpesvirus genes are classified into immediate-early (IE), early (E), or late (L) genes.
In all the RhCMV-based vectors made prior to our Ebola study, the heterologous target antigen has been place under a heterologous promoter that was expressed either at IE/E times or that has remained uncharacterized (this was also the case for other non-human primate herpesvirus-based vectors and MCMV). In contrast, the CMV vector used in our Ebola study was designed to place the Ebola virus GP under the control of the RhCMV endogenous pp65b promoter by replacement of the non-essential Rh112 (human CMV UL83 orthologue) gene with the Ebola virus GP gene. This places GP under control of the pp65 promoter which is expressed at L times. The use of this L promoter resulted in a complete reversal of the normal immune response associated with CMV and other herpesvirus-based vectors in the primate model. Specifically, it resulted in the immune response induced by the rhesus CMV vaccine being heavily biased towards induction of GP-specific antibodies, with minimal induction of GP-specific T cell responses.
This is a completely unexpected finding to those in the herpesvirus vaccine field based on the current understanding of these vaccines. Our idea concept, which has been reduced to practice in our recent study, is therefore that by utilizing promoters that express with different kinetics in herpesvirus-based vaccines we can now create vaccines that will bias the target pathogen-specific immune response towards either antibody production with diminished T cell responses (by use of a promoter expressed at L times), or towards T cell responses with diminished antibody responses (by use of a promoter expressed at IE and E times).
By use of promoters that are intermediate in their kinetics of expression (E-L), we would reasonably assume that a balanced antibody and T cell response would be achieved. Alternatively a mixture of vaccines using different kinetic classes of promoters to drive target antigen expression, either within distinct vaccines or within the same vaccine construct, would enable a balanced antibody and T cell response to be achieved. In this fashion, it will be possible to create vaccines that induce the desired balance of antibody and T cell responses against any target pathogen or cancer antigen.
The present invention is based on the principle of promoter usage to provide differential immune response modulation.
According to an aspect of the present invention there is provided a method of preparing a recombinant herpesvirus-based vector comprising the steps of: providing a nucleic acid sequence encoding a heterologous antigen; selecting a promoter for controlling the expression of the antigen, in which the promoter is selected to express at a time selected to provide a required immune response in a subject.
The present invention can be used to drive differential antibody and T cell response in a subject Selection of a temporal promoter can be used to drive a response in one direction or another, or to provide a balanced cell-mediated response.
According to a further the present invention there is provided a recombinant herpesvirus-based vector comprising a nucleic acid sequence encoding a heterologous antigen and a promoter for controlling the expression of the antigen, in which the promoter is expressed at a time selected to provide a required immune response in a subject.
The present invention may comprise a fully replication competent vector and/or an attenuated vector and/or a replication defective vector and/or any of the above vectors deleted or modified for immunomodulator genes.
The promoter may be selected to provide an immune response in a subject which is biased towards an antibody response in a subject.
The promoter may be selected to provide an immune response in a subject which is biased towards a T cell response in a subject.
The promoter may be expressed at E, IE or L times.
The herpesvirus-based vector may be selected from: Iltovirus, Proboscivirus, Cytomegalovirus, Mardivirus, Rhadinovirus, Macavirus, Roseolovirus, Simplexvirus, Scutavirus, Varicellovirus, Percavirus, Lymphocryptovirus, Muromegalovirus.
The herpesvirus-based vector may be a CMV-based vector.
The CMV-based vector may be selected from the non-exclusive list of human CMV, rhesus CMV, simian CMV, chimpanzee CMV, murine CMV and gorilla CMV.
The present invention also provides a recombinant herpesvirus-based vector comprising a nucleic acid sequence encoding a heterologous antigen and a promoter for controlling the expression of the antigen, in which the promoter is expressed at L times.
The present invention also provides a recombinant CMV-based vector comprising a nucleic acid sequence encoding a heterologous antigen and a promoter for controlling the expression of the antigen, in which the promoter is an endogenous CMV promoter that is expressed at L times, or a heterologous promoter expressed with comparable kinetics.
The present invention also provides a CMV-based vaccine with enhanced antibody production, comprising a recombinant CMV-based vector comprising a nucleic acid sequence encoding a heterologous antigen and a promoter for controlling the expression of the antigen, in which the promoter is expressed at L times.
The present invention also provides a vaccine comprising a recombinant herpesvirus-based vector, the vector construct comprising at least two nucleic acid sequences encoding heterologous antigens and each being under the control of a promoter, in which the promoter for each sequence is selected from a different kinetic class such that a predetermined balanced immune response can be achieved in a subject.
The present invention also provides a vaccine comprising a mixture of two or more types of recombinant herpesvirus-based vector, the vector types each comprising a nucleic acid sequence encoding a heterologous antigen and a promoter for controlling the expression of the antigen, in which the promoter in each type of vector is selected from a different kinetic class such that a predetermined balanced immune response can be achieved in a subject.
The heterologous antigen provided in aspects and embodiments of the present invention may be a pathogen-specific antigen or a tumor antigen, for example a human pathogen-specific antigen or a tumor antigen
The heterologous antigen may be a pathogen-specific antigen, for example a human pathogen-specific antigen
The pathogen from a (for example human) pathogen may be a viral antigen.
The antigen may be selected from a non-exclusive list of the following: human immunodeficiency virus, simian immuno-deficiency virus, Kaposi's sarcoma-associated herpesvirus, herpes simplex virus 1, herpes simplex virus 2, herpes virus B, Epstein Barr virus, hepatitis B virus, human papillomavirus, influenza virus, monkeypox virus, West Nile virus, Chikungunya virus, Ebola virus, hepatitis C virus, poliovirus, dengue virus, herpes virus B, Marburg virus, SARS virus, MERS virus.
In some aspects and embodiments a viral protein, an epitope or antigenic fragment thereof may be used as a heterologous antigen.
In some aspects and embodiments the pathogen-specific antigen may be a bacterial antigen.
In some aspects and embodiments the pathogen-specific antigen may be a fungal antigen.
In some aspects and embodiments the pathogen-specific antigen may be a protozoan antigen.
In some aspects and embodiments the pathogen-specific antigen may be a helminth antigen.
The vector/vaccine of the present invention may comprise, include or consist of one or more nucleic acid sequence as described and defined herein.
Genomes and sequence listings referred to and used in this study are:
The present invention also provides a composition comprising the vaccine or vector as described herein and a pharmaceutically acceptable carrier.
The present invention also provides a method of treating a subject with an infectious disease, or at risk of becoming infected with an infectious disease comprising selecting a subject in need of treatment and administering to the subject the recombinant vector or vaccine described herein or the composition described herein.
The present invention also provides for the use of the JQ795930 RhCMV vector or JQ795930 modified by additional attenuation as a vector for use in the treatment of prevention of disease in humans; for example, the use of this vector in a vaccine for the treatment of ebolavirus.
The present invention also provides a replication deficient HCMV-based vector including a human pp65 promoter controlling expression of ebolavirus glycoprotein. The vector may be derived from a diseased or a non-diseased source. Use of such a vector in the treatment or prevention of ebolavirus in humans is also provided.
The present invention also provides a replication deficient HCMV-based vector including a human EF-1α promoter controlling expression of ebolavirus glycoprotein. The vector may be derived from a diseased or a non-diseased source. Use of such a vector in the treatment or prevention of ebolavirus in humans is also provided.
The present invention also provides a method of treating a subject with a pre-existing infectious disease therapeutically, or at risk of becoming infected with an infectious disease prophylactically comprising selecting a subject in need of treatment and administering to the subject the recombinant vector or vaccine described herein or the composition described herein.
The present invention also provides a method of treating a subject with cancer, or at risk of developing cancer comprising selecting a subject in need of treatment and administering to the subject the recombinant vector or vaccine as described herein or the composition described herein.
The present invention also provides use of a vaccine, vector or composition as described herein for the prevention or treatment of a disease.
The present invention also provides a method of providing a modulated immune response comprising the steps of:
One aspect of the present invention relates to a CMV-based vaccine with enhanced antibody production provides nonhuman primates protection against ebolavirus.
The two independent 2014 ebolavirus (Zaire) (ZEBOV) outbreaks in West Africa and the Democratic Republic of the Congo (DRC) emphasize the public health problems filoviruses pose due to their lethality, unpredictable emergence, and localization to the poorest areas of the world1. Wildlife, primarily bats and great apes, play a critical role in filovirus transmission to humans by serving as reservoir or amplification species2-5. Interaction of humans with these species, either directly or by exposure to their habitat, has frequently been associated with human filovirus outbreaks3,6. ‘Self-disseminating’ cytomegalovirus (CMV)-based filovirus vaccines are one strategy to achieve filovirus-specific immunity in wildlife populations and block transmission to humans. Extending on earlier mouse studies7, rhesus CMV (RhCMV) expressing ZEBOV glycoprotein (GP) was shown to provide protective immunity from a highly lethal, low passage (7U) ZEBOV challenge. Surprisingly, the recombinant RhCMV GP-expressing ZEBOV vaccine induced high levels of GP-specific antibodies, but with minimal induction of GP-directed cellular immunity. We hypothesize that this bias towards humoral immunity targeting the heterologous target antigen, something not observed previously for RhCMV-based vectors, may result from novel promoter selection in the recombinant vaccine. In addition to showing substantial promise for development as a ‘self-disseminating’ vaccine to target ebolavirus in wild ape populations, this study identifies a potential strategy by which herpesvirus-based vectors can be rationally designed towards humoral, cellular or both arms of the adaptive immune response based on promoter selection.
The 2014 ZEBOV outbreak in the West African states of Guinea, Sierra Leone and Liberia is a stark example of the devastating effect of filoviruses following establishment of human-to-human transmission in densely populated areas with poor infrastructure1. Following introduction into the human population, ebolavirus is maintained by human-to-human transmission, and current interventional strategies are aimed at decreasing the reproductive number (R0) of transmission to R0<18. In previous outbreaks, this has been achieved by timely implementation of standard public health measures consisting of rapid identification and isolation of infected individuals, contact tracing and use of stringent barrier nursing procedure during patient care9. As evidenced in the West African outbreak, such interventional strategies may be insufficient to contain epidemic spread when delayed or under-resourced, especially following movement of ebolavirus into the urban environment of countries with inadequate public health systems. In the current outbreak, R0 fell from its early epidemic peak estimate of between 1.71 and 2.02, but as of Sep. 14, 2014 the net reproduction number Rt remains >1, and the number of cumulative confirmed and probable cases has been predicted to exceed 20,000 by early November1.
All human ebolavirus outbreaks are believed to result from zoonotic transmission from wild animal reservoir or transmission/amplification species3. Prevention of the initial zoonotic introduction from wildlife into the human population has therefore been proposed as an additional and complementary strategy to control human ebolavirus outbreaks3,7,10. Fruit bats have been identified as one probable reservoir for ebolavirus, and direct contact or exposure to environments inhabited and frequented by bats has been associated with human ebolavirus outbreaks3,6,11. Great apes (chimpanzees and western lowland gorillas) are regarded as a second main source of zoonotic ebolavirus transmission3,4,12,13, and vaccination of African great apes has been proposed as one possible strategy to prevent ebolavirus transmission to humans3,7,10. This approach may be especially suited to heavily under-resourced areas where the healthcare infrastructure is unable to control human-to-human transmission once ebolavirus infection has been established within urban populations. Ebolavirus is also highly lethal in African great apes2,4,12,14-16, and has resulted in a substantial reduction in the world gorilla population. Ebolavirus is therefore regarded as a major threat to the survival of chimpanzees and gorillas in the wild. In response to this threat, western lowland gorillas were upgraded to ‘Critically Endangered’ by the World Conservation Union in 200717. Vaccination of great apes is gaining support from primate conservationists due to its potential to stabilize ape populations against the devastating effects of ebolavirus.
Wild apes inhabiting geographically inaccessible tropical rainforests pose significant hurdles to conventional vaccination based on direct inoculation or baiting of individual animals. We recently proposed the use of a cytomegalovirus (CMV)-based ‘self-disseminating’ vaccine as one strategy to achieve the necessary vaccine coverage in these inaccessible and hostile environments7. In this scenario, high coverage would occur following animal-to-animal spread of the vaccine from a few initial directly inoculated ‘founder’ vaccines. CMV is a benign, species-specific member of the β-herpesvirus subfamily18,19. Due to its ability to induce substantial durable levels of T cells against heterologous encoded target antigens, CMV has gained considerable interest for development as a vaccine vector platform20-24. CMV is also able to spread easily through its host population, even in CMV-seropositive individuals7,25-28. In previous studies, we have shown the ability of a single dose of a murine CMV (MCMV) expressing a CD8 T cell epitope from nucleoprotein (NP) of ZEBOV (MCMV/ZEBOV-NPCTL) to induce durable, ZEBOV-specific CD8+ T cell immunity that remained protective against lethal ZEBOV challenge until >14 weeks post-vaccination (the latest time point measured in the studies).
Different aspects and embodiments of the invention may be used separately or together.
Further particular and preferred aspects of the present invention are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with the features of the independent claims as appropriate, and in combination other than those explicitly set out in the claims.
The present invention will now be more particularly described, by way of example, with reference to the above accompanying drawings, in which:
Supplemental Table 1. Combined Sanger and NGS Sequencing of BACs and reconstituted RhCMV/ZEBOV-GP (Clone 2-8 and 6-1).
Supplemental Table 2. Total and neutralizing antibody levels pre- and post-ZEBOV challenge.
Example embodiments are described below in sufficient detail to enable those of ordinary skill in the art to embody and implement the systems and processes herein described. It is important to understand that embodiments can be provided in many alternate forms and should not be construed as limited to the examples set forth herein.
Accordingly, while embodiments can be modified in various ways and take on various alternative forms, specific embodiments thereof are shown in the drawings and described in detail below as examples. There is no intent to limit to the particular forms disclosed. On the contrary, all modifications, equivalents, and alternatives falling within the scope of the appended claims should be included. Elements of the example embodiments are consistently denoted by the same reference numerals throughout the drawings and detailed description where appropriate.
The terminology used herein to describe embodiments is not intended to limit the scope. The articles “a,” “an,” and “the” are singular in that they have a single referent, however the use of the singular form in the present document should not preclude the presence of more than one referent. In other words, elements referred to in the singular can number one or more, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,” “comprising,” “includes,” and/or “including,” when used herein, specify the presence of stated features, items, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, items, steps, operations, elements, components, and/or groups thereof.
Unless otherwise defined, all terms (including technical and scientific terms) used herein are to be interpreted as is customary in the art. It will be further understood that terms in common usage should also be interpreted as is customary in the relevant art and not in an idealized or overly formal sense unless expressly so defined herein.
The aim of the current study was to assess the protective efficacy of a RhCMV-based ZEBOV vaccine expressing full length ZEBOV GP (RhCMV/ZEBOV-GP) against a low-passage lethal ZEBOV challenge in the macaque model, which is regarded as the ‘gold standard’ for translation of protection to great apes, including humans29. GP was chosen as the ZEBOV target, as this antigen has been shown to be a target of protective immunity when expressed from other vaccine platforms30,31. All RhCMV/ZEBOV-GP vectors were constructed using bacterial artificial chromosome (BAC)-based technology as previously described20,32,33. A schematic of RhCMV/ZEBOV-GP is shown in
A group of 4 Rhesus Monkeys (RMs) were inoculated with RhCMV/ZEBOV-GP in order to assess vaccine immunogenicity and efficacy (see schematic:
To determine whether immunity induced by RhCMV/ZEBOV-GP was able to protect animals from lethal ZEBOV disease, the RMs were then challenged with a lethal 1,000 ffu dose of low passage (7U) ZEBOV virus at day 0. RMs were monitored twice daily, and physical exams and blood draws were conducted on day 0, 4, 7, 14, 21, 28, and 35 post-ZEBOV challenge (
Given that the normal immunological phenotype for a RhCMV-based vector is heavily biased towards cellular ‘effector’ T cell memory with minimal antibody production20,32,33,36, the induction of high levels of anti-GP antibodies by the RhCMV/ZEBOV-GP, with an absence of detectable GP-specific T cells, was a surprising observation. Although the present study was not powered for identification of immune correlates of protection, the magnitude of total anti-GP IgG level corresponded to differences in protection between the RhCMV/ZEBOV-GP vaccinated animals [need to confirm day 0 level of RM#158], with the single non-protected vaccinated animal (RM#159) having consistently lower levels of total IgG against ZEBOV throughout the vaccine phase. Protection afforded by the recombinant vesicular stomatitis virus (VSV)-based ZEBOV vaccine currently undergoing Phase I human trials37 similarly corresponds to antibody level, with high levels of total anti-GP antibodies being a consistent predictor of survival38-40. A recent cellular depletion study in NHPs has confirmed that antibody rather than cellular GP-specific immune responses are of primary importance for rVSV-mediated protection41, The rapid ‘consumption’ of anti-GP antibodies that we observed in vaccinated animals at day 4 post-challenge (
The mechanism responsible for this unique antibody-biased immune response associated with RhCMV/ZEBOV-GP vaccination is an area of ongoing study. We hypothesize that the phenotype results from the expression of GP under control of the Rh112 L promoter, a promoter that is expressed a L times of RhCMV infection. Expression at this time coincides with the expression of multiple CMV-encoded immunoevasins that downregulate MHC expression. In our model, the inability to present the heterologous target antigen to T cells via the canonical MHC pathway would shift immune responses away from cellular towards humoral immunity. This also raises the possibility for rational design of vaccines that can direct the target pathogen-specific immune response towards either antibody production with diminished T cell responses (by use of a promoter expressed at L times), or towards T cell responses with diminished antibody responses (by use of a promoter expressed at IE and E times). By use of promoters that are intermediate in their kinetics of expression (E-L), a balanced antibody and T cell response could be achieved. Alternatively a mixture of vaccines using different kinetic classes of promoters to drive target antigen expression, either within distinct vaccines or within the same vaccine construct, would enable a balanced antibody and T cell response to be achieved.
In summary, the present study shows that the protective immunity of CMV vectors initially suggested in mouse studies using a MCMV-based vaccine translates into protective efficacy using RhCMV-based vectors in the ‘gold standard’ NHP ZEBOV challenge model. A primary goal of our studies is development of a ‘self-disseminating’ vaccine to target inaccessible African apes in the wild, both to prevent transmission of ebolavirus into the human population, but also to prevent ZEBOV-mediated eradication of these ape populations. Future studies will need to explore further the impact of CMV promoter usage on immune response characteristics, with the aim of inducing both cellular as well as substantial levels of humoral ZEBOV-specific immunity. In our study, RhCMV/ZEBOV-GP vaccine ‘take’ was clearly unperturbed by pre-existing immunity against the vector, as all RMs used in the present study were CMV seropositive at the time of vaccination. Immunity following animal-to-animal spread of the vector will now need to be investigated. Results from the present study also support the potential for development of CMV as prophylactic vaccine for ebolavirus in humans potentially using an attenuated or replication-deficient CMV platform. Ad-based and VSV-based ebolavirus vaccines differ in their modes of protection—the former being primarily associated with cellular immunity, whilst VSV is antibody-mediated. The current study suggests the unique possibility of being able to recruit both arms of the immune response together against ebolavirus infection in a single CMV vaccine by combined use of differential promoter usage.
Changes in various clinical parameters were measured over the duration of the study. (A) Kaplan-Meier survival curves, (B) temperature, (C) daily clinical scores, (D) viremia, (E) WBCs, (F) platelets, (G) AST levels, (H) ALT levels and (I) ALP levels.
Genomic characterization of RhCMV/ZEBOV-GP BAC. DNA from two independent clones of RhCMV/ZEBOV-GP [2-8 and 6-1] were digested with EcoRI followed by electrophoresis. The comparable digest pattern between RhCMV/ZEBOV-GP [6-1] and RhCMV/WT BAC shows the lack of any gross genomic rearrangement. A band shift was observed in the RhCMV/ZEBOV-GP BAC digest and was localized to X region.
Flow cytometry showing original dot-plot data presented graphically in
Fever was defined as ≧1° C. above baseline. Mild rash was defined as areas of petechiae covering less that 10% of the skin, moderate rash was defined as areas of petechiae covering 10-40% of the skin and severe rash was defined as areas of petechiae covering >40% of the skin. Leukocytopenia and thrombocytopenia were defined as a >30% decrease in numbers of WBCs and platelets, respectively. Leukocytosis and thrombocytosis were defined as a twofold or greater increase in numbers of WBCs and platelets above baseline levels, where WBC count >11,000. Elevated ALT, AST and ALP levels were defined as: ↑ (>2-fold; <4-fold increase), ↑↑ (>4-fold; <5-fold increase) and ↑↑↑ (>5-fold increase).
Combined Sanger and NGS Sequencing of BACs and reconstituted RhCMV/ZEBOV-GP (Clone 2-8 and 6-1).
Total and neutralizing antibody levels pre- and post-ZEBOV challenge.
Methods and any associated references are available in the online version of the paper at http://www.nature.com/nm/.
The study was approved by the Institutional Animal Care and Use Committee (IACUC) at the Rocky Mountain Laboratories (RML) (ASP#2014-020E). RML is accredited by the American Association for Accreditation of Laboratory Animal Care (Public Health Service/Office of Laboratory Animal Welfare Assurance #A4149-01). All animal work was performed in strict accordance with recommendations detailed in the ‘Guide for the Care and Use of Laboratory Animals’.42 Procedures were performed after ketamine-induced anesthesia by trained personnel under direct supervision of veterinarians. All efforts were made to minimize animal suffering based on recommendations of the working group report chaired by Sir David Weatherall “The use of non-human primates in research”.43 Rhesus macaques (Macaca mulatta) were housed under high containment at RML in individual adjoining primate cages enabling social interactions between animals. Humidity, temperature and light (12 hour light/dark cycles) were carefully regulated. Environmental enrichment was provided for animals by providing access to commercial toys. Animals were fed with commercial monkey chow, treats and fruit twice daily. Water was available ad libitum. Animals were monitored twice daily for the duration of the study (during both vaccine and ZEBOV challenge phases). Early endpoint criteria using score parameters approved by the RML IACUC were used to avoid unnecessary animal suffering (see below). Once the endstage of disease had been reached based on early endpoint criteria, animals were humanely euthanized.
We used a low passage ZEBOV (strain Mayinga) as the challenge virus for these studies44. ZEBOV was prepared in African green monkey kidney (Vero E6) cells. For stock production, we removed cellular debris by centrifugation, followed by aliquoting and storage of stocks under liquid nitrogen until use. EBOV utilizes transcriptional editing to regulate levels of soluble GP (sGP) compared to the transmembrane virion-associated peplomer form (GP1,2) by insertion of a non-templated adenine residue within a 7 uridine (poly-U) RNA-editing tract of the GP gene.45 Recombinant EBOVs containing a genomically-encoded 8U tract are also known to accumulate during EBOV passage in Vero E6 cells, but are rapidly selected against following in vivo passage.46,47 The ZEBOV challenge virus stock used in the present study was derived by limited in vitro passage in Vero E6 cells, and was confirmed to be primarily of the 7U form by reverse transcription (RT)-PCR followed by DNA Sanger sequencing. All infectious work using ZEBOV was performed under biosafety level 4 (BSL-4) containment at the Integrated Research Facility in the RML, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, Mont., USA.
We constructed RhCMV/ZEBOV-GP essentially as previously described48 by using E/T linear recombination to manipulate the parental RhCMV strain 68-1 genome cloned within a bacterial artificial chromosome (BAC) (designated pRhCMVIBAC-Cre).49 A previously constructed codon-optimized version of GP (optZGP) from ZEBOV (Mayinga strain 76; Accession number AF086833) was used as the target antigen34. The optZGP was codon-optimized by using the most frequent codons found in mammalian proteins, such that 70% (compared to 36% of non-optimized GP) of the codons present in optZGP are either the first or second most abundantly used mammalian codons.34 As a strategy to increase stability of optZGP expression, the optZGP open reading frame (ORF) was inserted within the RhCMV genome to replace the non-essential endogenous RhCMV Rh112 (pp65b) ORF (nucleotide positions 111,240 to 112,868 of RhCMV; see schematic FIG. X).50 This strategy places optZGP under control of the Rh112 promoter and also uses the endogenous Rh112 poly-adenylation signal sequence. Rh112 has been shown to be non-essential for infection or persistence of RhCMV in healthy seropositive or seronegative rhesus macaques.35 We also epitope tagged optZGP at the carboxy terminus with a V5 epitope tag to facilitate analysis of protein expression.51 The optZGP was cloned into a recombination cassette as a necessary requirement prior to E/T linear recombination. Within the recombination cassette, a FRT-flanked kanamycin resistance (KanR) marker was present immediately down-stream from optZGP ORF, which enabled selection of recombinant BAC clones on the basis of kanamycin resistance. We subsequently removed the KanR marker by arabinose induction of flp-recombinase, and recombinants were screened on the basis of kanamycin sensitivity. We analyzed recombinant RhCMV/ZEBOV-GP BAC clones by restriction enzyme digestion (
We used 6 captivity-bred adult male and female rhesus macaques of Indian genetic background. Animals were confirmed to be RhCMV seropositive by ELISA before initiation of the study (resulting from natural RhCMV infection). Animals were filovirus-naïve and were also free of cercopithicine herpesvirus 1, simian T-lymphotropic virus type 1, D-type simian retrovirus and simian immunodeficiency virus. We assigned animals to either RhCMV/ZEBOV-GP vaccine (n=4) or parental wild-type (WT) RhCMV control (n=2) groups with an aim of achieving a relatively equal distribution based on sex and age. On day −112, the vaccine group received a single sub-cutaneous (s.c.) bolus of a mixture of two independent clones of the RhCMV/ZEBOV-GP construct [5×106 plaque forming units (pfu)/construct]. The RhCMV WT control group received a single 1×107pfu s.c. inoculation of parental RhCMV WT (clone 68-1).49 Animals were boosted with either RhCMV/ZEBOV-GP or RhCMV WT at 20 week 12 (day −28). We collected blood samples at times indicated over the pre-challenge 112 day period (vaccine phase) (
We monitored animals twice daily over the entire study period (vaccine and ZEBOV challenge phases) using clinical score criteria approved by the RML IACUC. Assessment was based on the following criteria: i) general appearance, ii) condition of skin and fur, nose, mouth, eyes and head, iii) level of food intake, iv) quality and output of feces and urine, v) respiration, and vi) locomotor activity. Scores were recorded in a daily observation log, and animals were humanely euthanized when the total score reached 35. Euthanasia was also performed if any of the following signs were observed: i) impaired movement preventing access to food or water, ii) excessive weight loss, iii) loss of mental alertness, iv) difficulty in breathing, or v) prolonged inability to maintain upright posture.
We used a HemaVet® 950FS laser-based hematology analyzer (Drew Scientific) to analyze the following blood parameters in 20 μl volumes of EDTA-treated blood: i) total white blood cell count, ii) lymphocyte, platelet, reticulocyte and red blood cell counts, iii) hemoglobin, iv) hematocrit values, and v) mean corpuscular volume and hemoglobin concentrations. Serum chemistry was analyzed using a Piccolo Xpress Chemistry Analyzer using Piccolo General Chemistry 13 Panel discs (Abaxis).
We diluted rhesus macaque plasma samples 1:2 in serum matrix prior to analysis of plasma cytokine levels (IL-1Ra, IL-4, IL-6, IL-8, IL-12/23p40, IL-5, IL-17, soluble CD40L, IFNγ, MCP-1 and TNFα) by using Milliplex Non-Human Primate Magnetic Bead panels according to the manufacturer's (Millipore) protocol. Post-challenge plasma samples were inactivated by γ-irradiation (5 Mrad) prior to removal from BSL-4 containment under standard RML operating procedures as approved by the RML IBC.
We quantitated ZEBOV blood levels by using quantitative RT-PCR (qRT-PCR) as previously described.52 We also measured levels of infectious ZEBOV by using standard virus titration41, followed by calculation of 50% tissue culture infectious dose (TCID50) using the method of Reed and Muench.53 Tissues were homogenized prior to analysis.
Frequencies of CD4+ and CD8+ T cells directed against the ZEBOV (Mayinga) GP target antigen, as well as RhCMV immediate early 1 (IE1) protein were determined during the vaccine phase by intracellular cytokine staining (ICS) as previously described.41,54 For stimulation, PBMC (1-2×106 cells/well) were incubated in vitro with peptide pools (1 μg/ml final concentration) of overlapping peptides (11-mer with 5 amino acid overlap) representing each of the target ORFs. Incubation without antigen served as a background control. After 1 hour, brefeldin A (10 μg/ml) was added and cells were incubated for an additional 14 hours. Cells were surface stained using the following mAbs in indicated combinations: CD3, CD4 (eBioscience) and CD8β (Beckman Coulter). Cells were fixed and permeabilized according to manufacturer's recommendations (BioLegend) prior to staining for intracellular staining using mAbs against Ki67 (BD) and IFNγ and TNFα. Polychromatic flow cytometric analysis was performed on a LSR II (BD Biosciences), and data was analyzed by using FlowJo software (version 10; Tree Star, Inc.). Response frequencies were determined by subtracting background and then averaging background subtracted responses.
We measured total IgG antibody responses to RhCMV/ZEBOV-GP by ELISA using ZEBOV-GPΔ™ as a source of antigen, as previously described55. Analysis was performed as BSL-2. We show the end-point dilution titer (using a 4-fold dilution series). Analysis was performed at BSL-2. Post-challenge plasma samples were inactivated by γ-irradiation (5 Mrad) before removal from BSL-4 containment under standard RML operating procedures as approved by the RML Institutional Biosafety Committee (IBC). Samples were deemed positive when the OD value was higher than the mean plus 3 standard deviations of negative (RhCMV WT) sera.56
We analyzed plasma collected from animals at times indicated for ability to neutralize ZEBOV in an in vitro neutralization assay.34 Briefly, heat inactivated sera was serially diluted in DMEM, and then mixed 1:1 with ZEBOV expressing the EGFP reporter (200 FFU/well). After incubation at 37° C. for 60 minutes, we transferred 20 μl of the mixture onto subconfluent Vero E6 cells in a 96-well plate format and incubated for 30 minute at 37° C. Following addition of 1801 of DMEM supplemented with 1.5% carboxymethyl cellulose and 5% FBS, we cultured the cells for 4 days at 37° C. The cells were then washed with PBS and fixed in 10% neutral buffered formalin overnight under BSL-4 conditions. We then processed plates under BSL-4 conditions by conventional methods. We show the sera dilutions that resulted in 50% reduction in EGFP-positive cells following infection of Vero E6 cells with EGFP-labeled viruses.
Tests used for analysis are indicated in individual figure legends. One-way ANOVA with Bonferroni correction was used to compare immune responses and disease parameters between RhCMV/ZEBOV-GP and control groups. Statistical significance was determined at a level of 0.05. Kaplan-Meier log-rank test was used to compare survival rates between groups in ZEBOV challenge studies. All analysis was performed using Prism GraphPad Software (Version 5.0d).
We thank Dr P. Barry (University of California at Davis, Calif., USA) for providing the pRhCMV (68.1) RhCMV BAC, and Dr D. Court (NCI-Frederick, Md.) for providing the E/T-based recombination system, and, Dr T. Shenk and Dr W. Britt for providing antibodies.
Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiments shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1501523.3 | Jan 2015 | GB | national |
This application is a national stage entry under 35 U.S.C. 371 of International Application PCT/EP2016/051854 filed Jan. 28, 2016 (currently pending). International Application PCT/EP2016/051854 cites the priority of British Patent Application 1501523.3, filed on Jan. 30, 2015.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/051854 | 1/28/2016 | WO | 00 |
