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1.  Establishment of Fruit Bat Cells (Rousettus aegyptiacus) as a Model System for the Investigation of Filoviral Infection 
Background
The fruit bat species Rousettus aegyptiacus was identified as a potential reservoir for the highly pathogenic filovirus Marburg virus. To establish a basis for a molecular understanding of the biology of filoviruses in the reservoir host, we have adapted a set of molecular tools for investigation of filovirus replication in a recently developed cell line, R06E, derived from the species Rousettus aegyptiacus.
Methodology/Principal Findings
Upon infection with Ebola or Marburg viruses, R06E cells produced viral titers comparable to VeroE6 cells, as shown by TCID50 analysis. Electron microscopic analysis of infected cells revealed morphological signs of filovirus infection as described for human- and monkey-derived cell lines. Using R06E cells, we detected an unusually high amount of intracellular viral proteins, which correlated with the accumulation of high numbers of filoviral nucleocapsids in the cytoplasm. We established protocols to produce Marburg infectious virus-like particles from R06E cells, which were then used to infect naïve target cells to investigate primary transcription. This was not possible with other cell lines previously tested. Moreover, we established protocols to reliably rescue recombinant Marburg viruses from R06E cells.
Conclusion/Significance
These data indicated that R06E cells are highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir.
Author Summary
Marburg virus and several species of Ebola virus are endemic in central Africa and cause sporadic outbreaks in this region with mortality rates of up to 90%. So far, there is no vaccination or therapy available to protect people at risk in these regions. Recently, different fruit bats have been identified as potential reservoirs. One of them is Rousettus aegyptiacus. It seems that within huge bat populations only relatively small numbers are positive for filovirus-specific antibodies or filoviral RNA, a phenomenon that is currently not understood. As a first step towards understanding the biology of filoviruses in bats, we sought to establish a model system to investigate filovirus replication in cells derived from their natural reservoir. Here, we provide the first insights into this topic by monitoring filovirus infection of a Rousettus aegyptiacus derived cell line, R06E. We were able to show that filoviruses propagate well in R06E cells, which can, therefore, be used to investigate replication and transcription of filovirus RNA and to very efficiently perform rescue of recombinant Marburg virus using reverse genetics. These results emphasize the suitability of the newly established bat cell line for filovirus research.
doi:10.1371/journal.pntd.0000802
PMCID: PMC2927428  PMID: 20808767
2.  Vesicular Stomatitis Virus-Based Vaccines Protect Nonhuman Primates against Bundibugyo ebolavirus 
Ebola virus (EBOV) causes severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Currently, there are no licensed vaccines or therapeutics for human use. Recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode an EBOV glycoprotein in place of the VSV glycoprotein, have shown 100% efficacy against homologous Sudan ebolavirus (SEBOV) or Zaire ebolavirus (ZEBOV) challenge in NHPs. In addition, a single injection of a blend of three rVSV vectors completely protected NHPs against challenge with SEBOV, ZEBOV, the former Côte d'Ivoire ebolavirus, and Marburg virus. However, recent studies suggest that complete protection against the newly discovered Bundibugyo ebolavirus (BEBOV) using several different heterologous filovirus vaccines is more difficult and presents a new challenge. As BEBOV caused nearly 50% mortality in a recent outbreak any filovirus vaccine advanced for human use must be able to protect against this new species. Here, we evaluated several different strategies against BEBOV using rVSV-based vaccines. Groups of cynomolgus macaques were vaccinated with a single injection of a homologous BEBOV vaccine, a single injection of a blended heterologous vaccine (SEBOV/ZEBOV), or a prime-boost using heterologous SEBOV and ZEBOV vectors. Animals were challenged with BEBOV 29–36 days after initial vaccination. Macaques vaccinated with the homologous BEBOV vaccine or the prime-boost showed no overt signs of illness and survived challenge. In contrast, animals vaccinated with the heterologous blended vaccine and unvaccinated control animals developed severe clinical symptoms consistent with BEBOV infection with 2 of 3 animals in each group succumbing. These data show that complete protection against BEBOV will likely require incorporation of BEBOV glycoprotein into the vaccine or employment of a prime-boost regimen. Fortunately, our results demonstrate that heterologous rVSV-based filovirus vaccine vectors employed in the prime-boost approach can provide protection against BEBOV using an abbreviated regimen, which may have utility in outbreak settings.
Author Summary
Ebola viruses (EBOV), of which there are five species, are categorized as Category A Priority Pathogens and Tier 1 Select Agents by several US Government agencies as a result of their high mortality rates and potential for use as agents of bioterrorism. Currently, there are no vaccines or therapeutics approved for human use. Replication-competent, recombinant vesicular stomatitis virus (rVSV) vectors expressing filovirus glycoproteins (GP), in place of the VSV glycoprotein have shown promise in lethal nonhuman primate (NHP) models of filovirus infection as both single injection preventive vaccines and as post-exposure treatments. The recent outbreak of the fifth recognized EBOV species, Bundibugyo ebolavirus (BEBOV), demonstrates the need for vaccines that can be rapidly deployed to combat an outbreak of a new filovirus species. To date, rVSV-filovirus GP-based vaccines have only been able to protect against challenge with a homologous species of EBOV. Here, we show that the two heterologous rVSV-based filovirus vaccines available at the time of the original BEBOV outbreak can protect NHPs against BEBOV challenge using a short prime-boost vaccination strategy. While the prime-boost strategy was successful, a single injection blended vaccination strategy with the same vaccine vectors failed to provide protection. These data suggest that an abbreviated prime-boost regimen of 36 days may have utility for quickly responding to outbreaks caused by new species of EBOV.
doi:10.1371/journal.pntd.0002600
PMCID: PMC3868506  PMID: 24367715
3.  Comparison of Immunogenicity in Rhesus Macaques of Transmitted-Founder, HIV-1 Group M Consensus, and Trivalent Mosaic Envelope Vaccines Formulated as a DNA Prime, NYVAC, and Envelope Protein Boost 
Journal of Virology  2015;89(12):6462-6480.
ABSTRACT
An effective human immunodeficiency virus type 1 (HIV-1) vaccine must induce protective antibody responses, as well as CD4+ and CD8+ T cell responses, that can be effective despite extraordinary diversity of HIV-1. The consensus and mosaic immunogens are complete but artificial proteins, computationally designed to elicit immune responses with improved cross-reactive breadth, to attempt to overcome the challenge of global HIV diversity. In this study, we have compared the immunogenicity of a transmitted-founder (T/F) B clade Env (B.1059), a global group M consensus Env (Con-S), and a global trivalent mosaic Env protein in rhesus macaques. These antigens were delivered using a DNA prime-recombinant NYVAC (rNYVAC) vector and Env protein boost vaccination strategy. While Con-S Env was a single sequence, mosaic immunogens were a set of three Envs optimized to include the most common forms of potential T cell epitopes. Both Con-S and mosaic sequences retained common amino acids encompassed by both antibody and T cell epitopes and were central to globally circulating strains. Mosaics and Con-S Envs expressed as full-length proteins bound well to a number of neutralizing antibodies with discontinuous epitopes. Also, both consensus and mosaic immunogens induced significantly higher gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) responses than B.1059 immunogen. Immunization with these proteins, particularly Con-S, also induced significantly higher neutralizing antibodies to viruses than B.1059 Env, primarily to tier 1 viruses. Both Con-S and mosaics stimulated more potent CD8-T cell responses against heterologous Envs than did B.1059. Both antibody and cellular data from this study strengthen the concept of using in silico-designed centralized immunogens for global HIV-1 vaccine development strategies.
IMPORTANCE There is an increasing appreciation for the importance of vaccine-induced anti-Env antibody responses for preventing HIV-1 acquisition. This nonhuman primate study demonstrates that in silico-designed global HIV-1 immunogens, designed for a human clinical trial, are capable of eliciting not only T lymphocyte responses but also potent anti-Env antibody responses.
doi:10.1128/JVI.00383-15
PMCID: PMC4474309  PMID: 25855741
4.  Breadth of cellular and humoral immune responses elicited in rhesus monkeys by multi-valent mosaic and consensus immunogens 
Virology  2012;428(2):121-127.
To create an HIV-1 vaccine that generates sufficient breadth of immune recognition to protect against the genetically diverse forms of the circulating virus, we have been exploring vaccines based on consensus and mosaic protein designs. Increasing the valency of a mosaic immunogen cocktail increases epitope coverage but with diminishing returns, as increasingly rare epitopes are incorporated into the mosaic proteins. In this study we compared the immunogenicity of 2-valent and 3-valent HIV-1 envelope mosaic immunogens in rhesus monkeys. Immunizations with the 3-valent mosaic immunogens resulted in a modest increase in the breadth of vaccine-elicited T lymphocyte responses compared to the 2-valent mosaic immunogens. However, the 3-valent mosaic immunogens elicited significantly higher neutralizing responses to Tier 1 viruses than the 2-valent mosaic immunogens. These findings underscore the potential utility of polyvalent mosaic immunogens for eliciting both cellular and humoral immune responses to HIV-1.
doi:10.1016/j.virol.2012.03.012
PMCID: PMC3582221  PMID: 22521913
HIV-1 vaccine; Mosaic immunogen; T cell
5.  Mosaic Vaccines Elicit CD8+ T lymphocyte Responses in Monkeys that Confer Enhanced Immune Coverage of Diverse HIV Strains 
Nature medicine  2010;16(3):324-328.
An effective HIV vaccine must elicit immune responses that recognize genetically diverse viruses1, 2. It must generate CD8+ T lymphocytes that control HIV replication and CD4+ T lymphocytes that provide help for the generation and maintenance of both cellular and humoral immune responses against the virus3–5. Creating immunogens that can elicit cellular immune responses against the genetically varied circulating isolates of HIV presents an important challenge for creating an AIDS vaccine6, 7. Polyvalent mosaic immunogens derived by in silico recombination of natural strains of HIV are designed to induce cellular immune responses that recognize genetically diverse circulating virus isolates8. In the present study we immunized rhesus monkeys by plasmid DNA prime/ recombinant vaccinia virus boost using vaccine constructs expressing either consensus or polyvalent mosaic proteins. The mosaic immunogens elicited CD8+ T lymphocyte responses to more epitopes of each viral protein than the consensus immunogens, and to more variant sequences of CD8+ T lymphocyte epitopes. This increased breadth and depth of epitope recognition may contribute both to protection against infection by genetically diverse viruses and to the control of variant viruses that emerge as they mutate away from recognition by cytotoxic T lymphocytes.
doi:10.1038/nm.2108
PMCID: PMC2834806  PMID: 20173754
6.  Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs 
PLoS Pathogens  2015;11(6):e1005016.
The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel “wing” feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.
Author Summary
The filoviruses have caused multiple outbreaks among humans this decade, including a 90% lethal outbreak of Marburg virus in Angola and a significant, sustained outbreak of Ebola virus in West Africa. The viral surface glycoprotein (GP), which enables filoviruses to infect host cells, is the primary target of the immune system. Antibodies that target filovirus GP have been shown to provide life-saving therapy in nonhuman primates. However, the majority of known antibodies are only reactive against Ebola virus and not other emerging filoviruses. In this study, we present ten antibodies against Marburg virus, elicited by immunization of mice using engineered forms of its GP. Surprisingly, two antibodies exhibit some cross-reactivity to ebolaviruses (including species Ebola, Sudan, Bundibugyo, Reston). Other antibodies in this panel recognize a novel “wing” feature on a portion of GP that is unique to Marburg and does not exist in ebolaviruses, and protect 90%-100% of mice from lethal exposure. These antibodies, and their structural and functional analysis presented here, illuminate directions forward for therapeutics against Marburg virus.
doi:10.1371/journal.ppat.1005016
PMCID: PMC4482612  PMID: 26115029
7.  Broad Blockade Antibody Responses in Human Volunteers after Immunization with a Multivalent Norovirus VLP Candidate Vaccine: Immunological Analyses from a Phase I Clinical Trial 
PLoS Medicine  2015;12(3):e1001807.
Background
Human noroviruses (NoVs) are the primary cause of acute gastroenteritis and are characterized by antigenic variation between genogroups and genotypes and antigenic drift of strains within the predominant GII.4 genotype. In the context of this diversity, an effective NoV vaccine must elicit broadly protective immunity. We used an antibody (Ab) binding blockade assay to measure the potential cross-strain protection provided by a multivalent NoV virus-like particle (VLP) candidate vaccine in human volunteers.
Methods and Findings
Sera from ten human volunteers immunized with a multivalent NoV VLP vaccine (genotypes GI.1/GII.4) were analyzed for IgG and Ab blockade of VLP interaction with carbohydrate ligand, a potential correlate of protective immunity to NoV infection and illness. Immunization resulted in rapid rises in IgG and blockade Ab titers against both vaccine components and additional VLPs representing diverse strains and genotypes not represented in the vaccine. Importantly, vaccination induced blockade Ab to two novel GII.4 strains not in circulation at the time of vaccination or sample collection. GII.4 cross-reactive blockade Ab titers were more potent than responses against non-GII.4 VLPs, suggesting that previous exposure history to this dominant circulating genotype may impact the vaccine Ab response. Further, antigenic cartography indicated that vaccination preferentially activated preexisting Ab responses to epitopes associated with GII.4.1997. Study interpretations may be limited by the relevance of the surrogate neutralization assay and the number of immunized participants evaluated.
Conclusions
Vaccination with a multivalent NoV VLP vaccine induces a broadly blocking Ab response to multiple epitopes within vaccine and non-vaccine NoV strains and to novel antigenic variants not yet circulating at the time of vaccination. These data reveal new information about complex NoV immune responses to both natural exposure and to vaccination, and support the potential feasibility of an efficacious multivalent NoV VLP vaccine for future use in human populations.
Trial Registration
ClinicalTrials.gov NCT01168401
Lisa Lindesmith and colleagues assess the potential of a candidate virus-like particle (VLP) vaccine to induce antibody responses to antigenically divergent norovirus strains.
Editors' Summary
Background
Worldwide, noroviruses cause one in five cases of viral gastroenteritis (often called stomach flu or winter vomiting disease), the symptoms of which include nausea, vomiting, and diarrhea. There is no specific treatment for infection with these highly contagious viruses, and no established approach to vaccine development. While most people recover from the symptoms of norovirus infection within a few days, young children and the elderly may become severely ill or die. An estimated annual 300 million cases of norovirus infection contribute to roughly 260,000 deaths, mostly among this vulnerable demographic and mostly in low-income countries. Like influenza viruses, many noroviruses are evolving via a process known as antigenic drift. Antigens are components of infectious agents (including viruses) that are recognized by antibodies, proteins that bind to and neutralize foreign invaders. Over time, noroviruses develop small changes in their antigens that allow them to escape from antibodies produced in response to earlier infections. Every two to four years, because of accumulated antigenic drift, a new strain of norovirus emerges to which the human population has no direct antibody immunity, and an outbreak occurs. Because vaccines usually contain a component of the infectious agent that stimulates immunity, antigenic drift complicates the process of vaccine development. To be worth the cost and effort, a norovirus vaccine must confer immunity against a diverse range of norovirus strains, ideally including strains beyond those represented within the vaccine itself.
Partly because there is not a reliable method for growing noroviruses in the laboratory, recent efforts have focused on developing candidate vaccines using virus-like particles (VLPs). VLPs are constructed from laboratory-generated molecules of the virus’s capsid (outer shell). These capsid proteins self-assemble into icosahedral VLPs, which resemble the viral shell. VLPs cannot infect people or cause illness, but because they contain viral antigens, they can induce the immune system to produce antibodies that may neutralize actual viruses. VLPs can also be used to study the antibodies that people produce in response to vaccination or infection.
Why Was This Study Done?
VLP-based vaccines are relatively new, and their capacity to elicit a broad immune response conferring protection to an evolving range of norovirus strains is not established. One VLP vaccine based on a single strain that circulates primarily in children conferred immunity to that strain. Another, multivalent (containing a mix of VLPs from more than one strain) VLP vaccine elicited antibody generation, but in a phase I clinical trial did not confer immunity to infection by a strain that had previously circulated globally. In the current study, the researchers explored two key questions using laboratory analysis of blood samples drawn from participants in that trial. First, they tested whether the vaccine elicits antibody responses to a broad range of norovirus strains, as antibody responses can provide clues to the potential for this type of vaccine to confer broad immunity in the future. Second, they investigated how preexisting exposure to noroviruses affects the immune system’s response to a vaccine—strategic information that could aid in future vaccine development.
What Did the Researchers Do and Find?
The researchers tested serum (blood without cells or clotting proteins; serum contains the antibodies generated by the immune system) collected from ten participants receiving one injection of the VLP vaccine followed by a second injection 28 days later. They analyzed the serum specimens for antibodies to vaccine VLPs and also to VLPs representing viruses that were not contained in the vaccine. They used two methods, both utilizing VLPs generated from 11 norovirus strains: a traditional method that assesses binding of serum antibodies to each of these VLPs, and a more recent method that assays the ability of antibodies to block the interaction of each VLP with a molecule on intestinal cells that binds to the virus (the gut epithelial ligand), enabling norovirus to enter and infect cells. Prior studies suggest that this latter assay may be a better proxy for actual immunity.
The researchers’ major finding is that a multivalent VLP vaccine (two VLPs representing four strains of norovirus: one from a subgroup called genotype GI.1 and another consensus VLP of three strains from the subgroup GII.4) can rapidly elicit serum antibodies that bind a range of vaccine and non-vaccine VLPs, and that block binding of these VLPs to the gut epithelial ligand. Notably, vaccine recipients also generated antibodies reactive to two novel VLPs representing human noroviruses that they could not have previously encountered, indicating that prior exposure to each norovirus strain was not required for the full antibody response following vaccination. However, based on an analysis of which specific epitopes (small regions on an antigen) the population of antibodies binds, the authors report that antibody responses to the vaccine prominently target epitopes of a 1997 strain of human GII.4 norovirus, and propose that exposure history does influence the antibody response.
What Do these Findings Mean?
These findings raise the possibility that the VLP vaccine may induce immunity not only to norovirus strains that have caused past outbreaks, but also to variants that have yet to enter the population—a necessary attribute given the antigenic drift observed among noroviruses. The study also indicates that VLP-induced antibody responses to norovirus are consistent with the “antigenic seniority” model, in which strains to which an individual was previously exposed influence the binding properties of a vaccine-induced antibody population. This latter finding may influence the design of future norovirus vaccines.
These results must be interpreted cautiously, particularly as they pertain to the potential for a norovirus vaccine to protect against natural infection. The study is small, and antibody binding and blocking assays may not replicate how the immune system of a vaccine recipient will respond to true norovirus infection. Additionally, the study participants were all adults aged 18 to 49 years, while a vaccine is most needed for young children (who account for the majority of severe infections) and the elderly (who are most likely to die from infection). Unlike the study participants, young children lack preexisting antibodies to norovirus. Older people are more likely to have been previously exposed to norovirus, but may show attenuated immune responses to vaccination. Adapting to the different immune responses of these two groups remains a central challenge to norovirus vaccine development.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001807.
The World Health Organization provides a comprehensive description of the disease burden from diarrheal disease
The MedlinePlus encyclopedia has a page on viral gastroenteritis (in English and Spanish)
The US Centers for Disease Control and Prevention provides information on disease trends and outbreaks
The US Department of Health and Human Services offers guidance for prevention based on food safety
A 2014 interview with Academic Editor Benjamin Lopman explores the difficulty of developing a norovirus vaccine
The authors have previously published findings on the evolution of norovirus strains in PLOS Medicine and have discussed the challenges of norovirus therapeutic design in PLOS Pathogens
doi:10.1371/journal.pmed.1001807
PMCID: PMC4371888  PMID: 25803642
8.  Prospects for immunisation against Marburg and Ebola viruses 
Reviews in Medical Virology  2010;20(6):344-357.
SUMMARY
For more than 30 years the filoviruses, Marburg virus and Ebola virus, have been associated with periodic outbreaks of hemorrhagic fever that produce severe and often fatal disease. The filoviruses are endemic primarily in resource-poor regions in Central Africa and are also potential agents of bioterrorism. Although no vaccines or antiviral drugs for Marburg or Ebola are currently available, remarkable progress has been made over the last decade in developing candidate preventive vaccines against filoviruses in nonhuman primate models. Due to the generally remote locations of filovirus outbreaks, a single-injection vaccine is desirable. Among the prospective vaccines that have shown efficacy in nonhuman primate models of filoviral hemorrhagic fever, two candidates, one based on a replication-defective adenovirus serotype 5 and the other on a recombinant VSV (rVSV), were shown to provide complete protection to nonhuman primates when administered as a single injection. The rVSV-based vaccine has also shown utility when administered for postexposure prophylaxis against filovirus infections. A VSV-based Ebola vaccine was recently used to manage a potential laboratory exposure.
doi:10.1002/rmv.661
PMCID: PMC3394174  PMID: 20658513
9.  Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41 
PLoS Medicine  2008;5(1):e9.
Background
The generation of broadly neutralizing antibodies is a priority in the design of vaccines against HIV-1. Unfortunately, most antibodies to HIV-1 are narrow in their specificity, and a basic understanding of how to develop antibodies with broad neutralizing activity is needed. Designing methods to target antibodies to conserved HIV-1 epitopes may allow for the generation of broadly neutralizing antibodies and aid the global fight against AIDS by providing new approaches to block HIV-1 infection. Using a naturally occurring HIV-1 Envelope (Env) variant as a template, we sought to identify features of Env that would enhance exposure of conserved HIV-1 epitopes.
Methods and Findings
Within a cohort study of high-risk women in Mombasa, Kenya, we previously identified a subtype A HIV-1 Env variant in one participant that was unusually sensitive to neutralization. Using site-directed mutagenesis, the unusual neutralization sensitivity of this variant was mapped to two amino acid mutations within conserved sites in the transmembrane subunit (gp41) of the HIV-1 Env protein. These two mutations, when introduced into a neutralization-resistant variant from the same participant, resulted in 3- to >360-fold enhanced neutralization by monoclonal antibodies specific for conserved regions of both gp41 and the Env surface subunit, gp120, >780-fold enhanced neutralization by soluble CD4, and >35-fold enhanced neutralization by the antibodies found within a pool of plasmas from unrelated individuals. Enhanced neutralization sensitivity was not explained by differences in Env infectivity, Env concentration, Env shedding, or apparent differences in fusion kinetics. Furthermore, introduction of these mutations into unrelated viral Env sequences, including those from both another subtype A variant and a subtype B variant, resulted in enhanced neutralization susceptibility to gp41- and gp120-specific antibodies, and to plasma antibodies. This enhanced neutralization sensitivity exceeded 1,000-fold in several cases.
Conclusions
Two amino acid mutations within gp41 were identified that expose multiple discontinuous neutralization epitopes on diverse HIV-1 Env proteins. These exposed epitopes were shielded on the unmodified viral Env proteins, and several of the exposed epitopes encompass desired target regions for protective antibodies. Env proteins containing these modifications could act as a scaffold for presentation of such conserved domains, and may aid in developing methods to target antibodies to such regions.
Julie Overbaugh and colleagues analyze an HIV strain with high susceptibility to antibody neutralization and identify two gp41 envelope mutations that confer this sensitivity by exposing multiple neutralization epitopes.
Editors' Summary
Background.
In 1984 when scientists identified human immunodeficiency virus (HIV)—the cause of acquired immunodeficiency syndrome (AIDS)—many experts believed that a vaccine against HIV infection would soon be developed. Nearly 25 years later, there is still no such vaccine and with about 2.5 million new HIV infections in 2007, an effective vaccine is urgently needed to contain the AIDS epidemic. Vaccines provide protection against infectious diseases by priming the immune system to deal quickly and effectively with viruses and other pathogens. Vaccines do this by exposing the immune system to an immunogen—a fragment or harmless version of the pathogen. The immune system mounts a response against the immunogen and also “learns” from this experience so that if it is ever challenged with a virulent version of the same pathogen, it can quickly contain the threat. Many vaccines work by stimulating an antibody response. Antibodies are proteins made by the immune system that bind to molecules called antigens on the surface of pathogens. Antibodies that inactivate the invader upon binding to it are called “neutralizing” antibodies.
Why Was This Study Done?
Several characteristics of HIV have hampered the development of an effective vaccine. An “envelope” protein consisting of two subunits called gp120 and gp41 covers the outside of HIV. Many regions of this protein change rapidly, so the antibody response stimulated by a vaccine containing the envelope protein of one HIV variant provides little protection against other variants. However, other regions of the protein rarely change, so a vaccine that stimulates the production of antibodies to these “conserved” regions is likely to provide protection against many HIV variants. That is, it will stimulate the production of broadly neutralizing antibodies. Unfortunately, it has been difficult to find HIV vaccines that do this, because these conserved regions are often hidden from the immune system by other parts of the envelope protein. In this study, the researchers investigate the envelope protein of an HIV-1 variant they have isolated that is highly susceptible to inactivation by antibodies specific for these conserved regions. Comparing the envelope protein of this sensitive virus to closely related envelope proteins that are resistant to neutralization could identify features that might, if included in an envelope protein immunogen, produce a vaccine capable of generating broadly neutralizing antibodies.
What Did the Researchers Do and Find?
The researchers isolated a subtype A HIV-1 variant from a newly infected woman in Kenya that was efficiently neutralized by monoclonal antibodies (antibodies made by cells that have been cloned in the laboratory). These antibodies were specific for several different conserved regions of gp41 and gp120. The isolate was also neutralized by antibodies in blood from HIV-1-infected people. The envelope protein of the sensitive variant was the same as that of a resistant variant isolated at the same time from the woman, except for four amino acid changes in conserved regions of gp41 (proteins are made from long strings of amino acids). Using a technique called site-directed mutagenesis, the researchers introduced these amino acid changes into envelope proteins made in the laboratory and determined that just two of these changes were responsible for the neutralization sensitivity of the HIV-1 variant. The introduction of these two changes into the neutralization resistant variant and into the unrelated envelope sequences of another subtype A (common in Africa) HIV-1 variant and a subtype B HIV-1 (common in Europe and the Western Hemisphere) variant increased the sensitivity of all these viruses to antibody neutralization.
What Do These Findings Mean?
These findings show that two amino acid changes in gp41 of a neutralization-sensitive HIV-1 variant are responsible for the sensitivity of this variant to several neutralizing antibodies. The finding that the inclusion of these changes in the envelope protein of neutralization-resistant HIV-1 variants greatly increases their sensitivity to neutralizing antibodies indicates that the normally shielded regions of the protein are somehow made accessible to antibody by these changes. One possibility is that the amino acid changes might modify the overall shape of the envelope protein, thus exposing multiple, normally hidden regions in the HIV-1 envelope protein to antibodies. Importantly, these findings open up the possibility that the inclusion of these modifications in envelope-based immunogens might improve the ability of vaccines to generate broadly neutralizing antibodies against HIV-1.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050009.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIVInSite has comprehensive information on all aspects of HIV/AIDS, including links to resources dealing with HIV vaccine development
Information is available from Avert, an international AIDS charity, on all aspects of HIV and AIDS, including HIV vaccines
The US Centers for Disease Control and prevention provides information on HIV/AIDS including information on its HIV vaccine unit (in English and some information in Spanish)
The AIDS Vaccine Clearinghouse provides clear information about HIV vaccine science, research and product development
The International AIDS Vaccine Initiative also provides straightforward information about the development of HIV vaccines
doi:10.1371/journal.pmed.0050009
PMCID: PMC2174964  PMID: 18177204
10.  Filoviruses are ancient and integrated into mammalian genomes 
Background
Hemorrhagic diseases from Ebolavirus and Marburgvirus (Filoviridae) infections can be dangerous to humans because of high fatality rates and a lack of effective treatments or vaccine. Although there is evidence that wild mammals are infected by filoviruses, the biology of host-filovirus systems is notoriously poorly understood. Specifically, identifying potential reservoir species with the expected long-term coevolutionary history of filovirus infections has been intractable. Integrated elements of filoviruses could indicate a coevolutionary history with a mammalian reservoir, but integration of nonretroviral RNA viruses is thought to be nonexistent or rare for mammalian viruses (such as filoviruses) that lack reverse transcriptase and replication inside the nucleus. Here, we provide direct evidence of integrated filovirus-like elements in mammalian genomes by sequencing across host-virus gene boundaries and carrying out phylogenetic analyses. Further we test for an association between candidate reservoir status and the integration of filoviral elements and assess the previous age estimate for filoviruses of less than 10,000 years.
Results
Phylogenetic and sequencing evidence from gene boundaries was consistent with integration of filoviruses in mammalian genomes. We detected integrated filovirus-like elements in the genomes of bats, rodents, shrews, tenrecs and marsupials. Moreover, some filovirus-like elements were transcribed and the detected mammalian elements were homologous to a fragment of the filovirus genome whose expression is known to interfere with the assembly of Ebolavirus. The phylogenetic evidence strongly indicated that the direction of transfer was from virus to mammal. Eutherians other than bats, rodents, and insectivores (i.e., the candidate reservoir taxa for filoviruses) were significantly underrepresented in the taxa with detected integrated filovirus-like elements. The existence of orthologous filovirus-like elements shared among mammalian genera whose divergence dates have been estimated suggests that filoviruses are at least tens of millions of years old.
Conclusions
Our findings indicate that filovirus infections have been recorded as paleoviral elements in the genomes of small mammals despite extranuclear replication and a requirement for cooption of reverse transcriptase. Our results show that the mammal-filovirus association is ancient and has resulted in candidates for functional gene products (RNA or protein).
doi:10.1186/1471-2148-10-193
PMCID: PMC2906475  PMID: 20569424
11.  Mosaic HIV-1 Gag antigens can be Processed and Presented to human HIV-specific CD8+ T cells 
Polyvalent “mosaic” HIV immunogens offer a potential solution for generating vaccines that can elicit immune responses against genetically diverse viruses. However, it is unclear whether key T cell epitopes can be processed and presented from these synthetic antigens and recognized by epitope-specific human T cells. Here we tested the ability of mosaic HIV immunogens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors to process and present major HIV clade B and clade C CD8 T cell epitopes in human cells. A bivalent mosaic vaccine expressing HIV Gag sequences was used to transduce PBMC from 12 HIV-1-infected individuals from the US and 10 HIV-1-infected individuals from South Africa, and intracellular cytokine staining together with tetramer staining was used to assess the ability of mosaic Gag antigens to stimulate pre-existing memory responses compared to natural clade B and C vectors. Mosaic Gag antigens expressed all 8 clade B epitopes tested in 12 US subjects and all 5 clade C epitopes tested in 10 South African subjects. Overall, the magnitude of cytokine production induced by stimulation with mosaic antigens was comparable to clade B and clade C antigens tested, but the mosaic antigens elicited greater cross-clade recognition. Additionally, mosaic antigens also induced HIV-specific CD4 T cell responses. Our studies demonstrate that mosaic antigens express major clade B and clade C viral T cell epitopes in human cells, and support the evaluation of mosaic HIV-1 vaccines in humans.
doi:10.4049/jimmunol.1004231
PMCID: PMC3129742  PMID: 21576505
12.  Expanded Breadth of the T-Cell Response to Mosaic Human Immunodeficiency Virus Type 1 Envelope DNA Vaccination▿ †  
Journal of Virology  2008;83(5):2201-2215.
An effective AIDS vaccine must control highly diverse circulating strains of human immunodeficiency virus type 1 (HIV-1). Among HIV-1 gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV-1 Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential T-cell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. One-, two-, and three-mosaic sets that increased theoretical epitope coverage were developed. The breadth and magnitude of T-cell immunity stimulated by these vaccines were compared to those for natural strain Envs; additional comparisons were performed on mutant Envs, including gp160 or gp145 with or without V regions and gp41 deletions. Among them, the two- or three-mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the three-mosaic set elicited responses to an average of eight peptide pools, compared to two pools for a set of three natural Envs. Synthetic mosaic HIV-1 antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based HIV-1 vaccines.
doi:10.1128/JVI.02256-08
PMCID: PMC2643734  PMID: 19109395
13.  A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease 
PLoS Pathogens  2009;5(12):e1000695.
Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.
Author Summary
Arenaviruses cause significant morbidity and mortality worldwide and are also regarded as a potential bioterrorist threat. CD8+ T cells restricted by class I MHC molecules clearly play a protective role in murine models of arenavirus infection, yet little is known about the epitopes recognized in the context of human class I MHC (HLA). Here, we defined 20 CD8+ T cell epitopes restricted by HLA class I molecules, derived from 7 different species of arenaviruses associated with human disease. To accomplish this task, we utilized epitope predictions, in vitro HLA binding assays, and HLA transgenic mice inoculated with recombinant vaccinia viruses (rVACV) expressing arenavirus antigens. Because our analysis targeted two of the most common HLA types worldwide, we project that the CD8+ T cell epitope set provides broad coverage against diverse ethnic groups within the human population. Furthermore, we show that immunization with a cocktail of these epitopes protects HLA transgenic mice from challenge with rVACV expressing antigens from different arenavirus species. Our findings suggest that a cell-mediated vaccine strategy might be able to protect against infection mediated by multiple arenavirus species.
doi:10.1371/journal.ppat.1000695
PMCID: PMC2787016  PMID: 20019801
14.  Analysis of the human env-specific cytotoxic T-lymphocyte (CTL) response in natural human immunodeficiency virus type 1 infection: low prevalence of broadly cross-reactive env-specific CTL. 
Journal of Virology  1996;70(12):8468-8476.
Major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to persistent virus infections. Candidate vaccines against human immunodeficiency virus type 1 (HIV-1) should elicit broad cross-reactive immunity to confer protection against different strains of HIV-1. As it is likely that candidate vaccines will include the envelope gene product Env, we determined the proportion of CTL clones which recognized variable and conserved determinants in three env variants during natural infection. Limiting dilution analysis was used to characterize numerous short-term CTL clones derived from peripheral blood of HIV-1-infected subjects, using split-well analysis to assay cytotoxicity against target cells expressing gp160env of HIV-1 strains IIIB, MN, and RF. In 9 of 12 HIV-1-infected subjects, at the clonal level most env-specific CTL recognized determinant(s) within one env variant but not in the other variants. In some subjects, CTL recognized multiple nonconserved determinants in different variants. The pattern of recognition of different env variants was relatively stable over time. In most of the patients studied, the proportion of CTL which showed cross-recognition of conserved determinants shared among the three strains was low. Two novel CTL epitopes within gp41 were identified by using 15-mer peptides of the HIV-SF2 sequence. When specific peptide was used to stimulate CTL precursors in vitro, the frequency of peptide-specific CTL precursors was very high, but the CTL elicited by this stimulation were highly strain specific. We conclude that the use of a single HIV env variant to detect CTL activity can underestimate the magnitude and complexity of the env-specific CTL response. The low prevalence of CTL clones which show cross-recognition of conserved determinants may have implications for immunization strategies based solely on env; to elicit broadly cross-reactive CTL other, more conserved viral antigens are likely to be needed in addition to env. Because of its capacity to distinguish CTL responses against different virus strains, limiting dilution analysis is particularly appropriate to quantitate the immune responses generated by candidate env-based vaccines.
PMCID: PMC190937  PMID: 8970969
15.  Defining epitope coverage requirements for T cell-based HIV vaccines: Theoretical considerations and practical applications 
Background
HIV vaccine development must address the genetic diversity and plasticity of the virus that permits the presentation of diverse genetic forms to the immune system and subsequent escape from immune pressure. Assessment of potential HIV strain coverage by candidate T cell-based vaccines (whether natural sequence or computationally optimized products) is now a critical component in interpreting candidate vaccine suitability.
Methods
We have utilized an N-mer identity algorithm to represent T cell epitopes and explore potential coverage of the global HIV pandemic using natural sequences derived from candidate HIV vaccines. Breadth (the number of T cell epitopes generated) and depth (the variant coverage within a T cell epitope) analyses have been incorporated into the model to explore vaccine coverage requirements in terms of the number of discrete T cell epitopes generated.
Results
We show that when multiple epitope generation by a vaccine product is considered a far more nuanced appraisal of the potential HIV strain coverage of the vaccine product emerges. By considering epitope breadth and depth several important observations were made: (1) epitope breadth requirements to reach particular levels of vaccine coverage, even for natural sequence-based vaccine products is not necessarily an intractable problem for the immune system; (2) increasing the valency (number of T cell epitope variants present) of vaccine products dramatically decreases the epitope requirements to reach particular coverage levels for any epidemic; (3) considering multiple-hit models (more than one exact epitope match with an incoming HIV strain) places a significantly higher requirement upon epitope breadth in order to reach a given level of coverage, to the point where low valency natural sequence based products would not practically be able to generate sufficient epitopes.
Conclusions
When HIV vaccine sequences are compared against datasets of potential incoming viruses important metrics such as the minimum epitope count required to reach a desired level of coverage can be easily calculated. We propose that such analyses can be applied early in the planning stages and during the execution phase of a vaccine trial to explore theoretical and empirical suitability of a vaccine product to a particular epidemic setting.
doi:10.1186/1479-5876-9-212
PMCID: PMC3284408  PMID: 22152192
16.  Induction of a major histocompatibility complex class I-restricted cytotoxic T-lymphocyte response to a highly conserved region of human immunodeficiency virus type 1 (HIV-1) gp120 in seronegative humans immunized with a candidate HIV-1 vaccine. 
Journal of Virology  1994;68(5):3145-3153.
Efforts to induce broadly reactive immunity against human immunodeficiency virus type 1 (HIV-1) have been impaired by the extent of sequence variation exhibited by this lentivirus. Cytotoxic T lymphocytes (CTL) specific for other viruses such as influenza virus have been shown to mediate immunity against divergent viral strains, a property that is related to the ability of CTL to recognize processed antigen derived from conserved viral proteins. A recent candidate HIV-1 vaccine regimen has been described in which subjects receive a primary immunization with a recombinant vaccinia virus expressing gp160 and then a booster immunization with recombinant gp160. Volunteers immunized with this regimen have exhibited augmented humoral responses and have also developed CD4+ and CD8+ CTL specific for gp160. In this report, we have identified the epitopes recognized by CD4+ and CD8+ CTL obtained from two vaccines. An immunodominant CD8+ CTL response was HLA-A3.1 restricted and recognized a 10-amino-acid epitope (gp120/38-47) in a highly conserved region of gp120. CTL specific for the epitope gp120/38-47 were able to lyse targets sensitized with peptides corresponding to all known natural sequence variants in this region. In addition, other HLA class I-restricted CTL epitopes were identified in relatively conserved regions of gp120 and gp41, and CD4+ CTL were shown to recognize two different regions of gp120. Thus, in these two volunteers, immunization with a single strain of HIV-1 induced CD4+ and CD8+ CTL that are specific for multiple conserved regions of HIV-1 and would be expected to recognize a broad range of viral isolates.
PMCID: PMC236805  PMID: 7908700
17.  Ad35 and Ad26 Vaccine Vectors Induce Potent and Cross-Reactive Antibody and T-Cell Responses to Multiple Filovirus Species 
PLoS ONE  2012;7(12):e44115.
Filoviruses cause sporadic but highly lethal outbreaks of hemorrhagic fever in Africa in the human population. Currently, no drug or vaccine is available for treatment or prevention. A previous study with a vaccine candidate based on the low seroprevalent adenoviruses 26 and 35 (Ad26 and Ad35) was shown to provide protection against homologous Ebola Zaire challenge in non human primates (NHP) if applied in a prime-boost regimen. Here we have aimed to expand this principle to construct and evaluate Ad26 and Ad35 vectors for development of a vaccine to provide universal filovirus protection against all highly lethal strains that have caused major outbreaks in the past. We have therefore performed a phylogenetic analysis of filovirus glycoproteins to select the glycoproteins from two Ebola species (Ebola Zaire and Ebola Sudan/Gulu,), two Marburg strains (Marburg Angola and Marburg Ravn) and added the more distant non-lethal Ebola Ivory Coast species for broadest coverage. Ad26 and Ad35 vectors expressing these five filovirus glycoproteins were evaluated to induce a potent cellular and humoral immune response in mice. All adenoviral vectors induced a humoral immune response after single vaccination in a dose dependent manner that was cross-reactive within the Ebola and Marburg lineages. In addition, both strain-specific as well as cross-reactive T cell responses could be detected. A heterologous Ad26–Ad35 prime-boost regime enhanced mainly the humoral and to a lower extend the cellular immune response against the transgene. Combination of the five selected filovirus glycoproteins in one multivalent vaccine potentially elicits protective immunity in man against all major filovirus strains that have caused lethal outbreaks in the last 20 years.
doi:10.1371/journal.pone.0044115
PMCID: PMC3516506  PMID: 23236343
18.  Short Conserved Sequences of HIV-1 Are Highly Immunogenic and Shift Immunodominance 
Journal of Virology  2014;89(2):1195-1204.
ABSTRACT
Cellular immunity is pivotal in HIV-1 pathogenesis but is hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore, we selected four relatively conserved regions (Gag amino acids 148 to 214 and 250 to 335, Env amino acids 521 to 606, and Nef amino acids 106 to 148), each created in three mosaics, to provide better coverage of M-group HIV-1 sequences. A conserved-region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics each (each region at a separate injection site) was compared to a whole-protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus type 5 boost (weeks 0, 4, 8, and 24, respectively). Although CRV inserts were about one-fifth that of WPV, the CRV generated comparable-magnitude blood CD4+ and CD8+ T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions and the outside regions. The CRV yielded a conserved-region targeting density that was approximately 5-fold higher than that of the WPV. A similar pattern was seen for bronchoalveolar lymphocytes, but with quadruple the magnitudes seen in blood. Overall, these findings demonstrate that the selected conserved regions are highly immunogenic and that anatomically isolated vaccinations with these regions focus immunodominance compared to the case for full-length protein vaccination.
IMPORTANCE HIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one-fifth of the whole Gag/Env/Nef sequence, the vaccines elicited equivalent total magnitudes of both CD4+ and CD8+ T lymphocyte responses. These data demonstrate the immunogenicity of these small conserved regions and the potential for a vaccine to steer immunodominance toward conserved epitopes.
doi:10.1128/JVI.02370-14
PMCID: PMC4300636  PMID: 25378501
19.  Identification and characterization of merozoite surface protein 1 epitope 
Bioinformation  2009;4(1):1-5.
Malaria is an important tropical infection which urgently requires intervention of an effective vaccine. Antigenic variations of the parasite and allelic diversity of the host are main problems in the development of an effective malaria vaccine. Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum­derived antigens are shown to play an important role for the protection against malaria. The merozoite surface protein 1 (MSP1) is expressed in all the four life-cycle stages of Plasmodium falciparum and did not find any sequence similarity to human and mouse reference proteins. MSP1 is a known target of the immune response and a single CTL epitope binding to the HLA­A*0201 is available for merozoite form. Here, we report the results from the computational characterization of MSP1, precursor (1720 residue) and screening of highest scoring potential CTL epitopes for 1712 overlapping peptides binding to thirty four HLA class­I alleles and twelve HLA class­I supertypes (5 HLA­A and 7 HLA­B) using bioinformatics tools. Supertypes are the clustered groups of HLA class­I molecules, representing a sets of molecules that share largely overlapping peptide binding specificity. The prediction results for MSP1 as adhesin and adhesin-like in terms of probability is 1.0. Results also show that MSP1 has orthologs to other related species as well as having non allergenicity and single transmembrane properties demonstrating its suitability as a vaccine candidate. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.
PMCID: PMC2770364  PMID: 20011145
epitope; supertype; vaccine; malaria; bioinformatics
20.  Demonstration of Cross-Protective Vaccine Immunity against an Emerging Pathogenic Ebolavirus Species 
PLoS Pathogens  2010;6(5):e1000904.
A major challenge in developing vaccines for emerging pathogens is their continued evolution and ability to escape human immunity. Therefore, an important goal of vaccine research is to advance vaccine candidates with sufficient breadth to respond to new outbreaks of previously undetected viruses. Ebolavirus (EBOV) vaccines have demonstrated protection against EBOV infection in nonhuman primates (NHP) and show promise in human clinical trials but immune protection occurs only with vaccines whose antigens are matched to the infectious challenge species. A 2007 hemorrhagic fever outbreak in Uganda demonstrated the existence of a new EBOV species, Bundibugyo (BEBOV), that differed from viruses covered by current vaccine candidates by up to 43% in genome sequence. To address the question of whether cross-protective immunity can be generated against this novel species, cynomolgus macaques were immunized with DNA/rAd5 vaccines expressing ZEBOV and SEBOV glycoprotein (GP) prior to lethal challenge with BEBOV. Vaccinated subjects developed robust, antigen-specific humoral and cellular immune responses against the GP from ZEBOV as well as cellular immunity against BEBOV GP, and immunized macaques were uniformly protected against lethal challenge with BEBOV. This report provides the first demonstration of vaccine-induced protective immunity against challenge with a heterologous EBOV species, and shows that Ebola vaccines capable of eliciting potent cellular immunity may provide the best strategy for eliciting cross-protection against newly emerging heterologous EBOV species.
Author Summary
Ebola virus causes death, fear, and economic disruption during outbreaks. It is a concern worldwide as a natural pathogen and a bioterrorism agent, and has caused death to residents and tourists of Africa where the virus circulates. A vaccine strategy to protect against all circulating Ebola viruses is complicated by the fact that there are five different virus species, and individual vaccines provide protection only against those included in the vaccine. Making broad vaccines that contain multiple components is complicated, expensive, and poses challenges for regulatory approval. Therefore, in the present work, we examined whether a prime-boost immunization strategy with a vaccine targeted to one Ebola virus species could cross protect against a different species. We found that genetic immunization with vectors expressing the Ebola virus glycoprotein from Zaire blocked infection with a newly emerged virus species, Bundibugyo EBOV, not represented in the vaccine. Protection occurred in the absence of antibodies against the second species and was mediated instead by cellular immune responses. Therefore, single-component vaccines may be improved to protect against multiple Ebola viruses if they are designed to generate this type of immunity.
doi:10.1371/journal.ppat.1000904
PMCID: PMC2873919  PMID: 20502688
21.  Challenges in the Design of a T Cell Vaccine in the Context of HIV-1 Diversity 
Viruses  2014;6(10):3968-3990.
The extraordinary variability of HIV-1 poses a major obstacle to vaccine development. The effectiveness of a vaccine is likely to vary dramatically in different populations infected with different HIV-1 subtypes, unless innovative vaccine immunogens are developed to protect against the range of HIV-1 diversity. Immunogen design for stimulating neutralizing antibody responses focuses on “breadth” – the targeting of a handful of highly conserved neutralizing determinants on the HIV-1 Envelope protein that can recognize the majority of viruses across all HIV-1 subtypes. An effective vaccine will likely require the generation of both broadly cross-neutralizing antibodies and non-neutralizing antibodies, as well as broadly cross-reactive T cells. Several approaches have been taken to design such broadly-reactive and cross-protective T cell immunogens. Artificial sequences have been designed that reduce the genetic distance between a vaccine strain and contemporary circulating viruses; “mosaic” immunogens extend this concept to contain multiple potential T cell epitope (PTE) variants; and further efforts attempt to focus T cell immunity on highly conserved regions of the HIV-1 genome. Thus far, a number of pre-clinical and early clinical studies have been performed assessing these new immunogens. In this review, the potential use of these new immunogens is explored.
doi:10.3390/v6103968
PMCID: PMC4213573  PMID: 25341662
HIV-1; diversity; broadly cross-reactive T cell responses; vaccine; immunogen design
22.  Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum sporozoite surface protein 2 
The Journal of Experimental Medicine  1995;182(5):1435-1445.
Vaccines designed to protect against malaria by inducing CD8+ cytotoxic T lymphocytes (CTL) in individuals of diverse HLA backgrounds must contain multiple conserved epitopes from various preerythrocytic-stage antigens. Plasmodium falciparum sporozoite surface protein 2 (PfSSP2) is considered an important antigen for inclusion in such vaccines, because CD8+ CTL against the P. yoelii SSP2 protect mice against malaria by eliminating infected hepatocytes. To develop PfSSP2 as a component of malaria vaccines, we investigated the presence of anti- PfSSP2 CTL in two HLA-B8+ volunteers immunized with irradiated P. falciparum sporozoites and characterized their CTL responses using PfSSP2-derived 15-amino acid peptides bearing the HLA-B8-binding motif. Peripheral blood mononuclear cells from both volunteers stimulated with recombinant vaccinia expressing PfSSP2 displayed antigen-specific, genetically restricted, CD8+ T cell-dependent CTL activity against autologous target cells expressing PfSSP2. Of the five HLA-B8 motif- bearing 15-mers identified in the PfSSP2 sequence, two peptides sharing a 10-amino acid overlap sensitized HLA-B8-matched target cells from both volunteers for lysis by peptide-stimulated effectors. The CTL activity was HLA-B8 restricted and dependent on CD8+ T cells. Analysis of the three shorter peptides representing HLA-B8 motif-bearing sequences within the two positive peptides for their ability to bind to HLA-B8 in vitro, and to sensitize target cells for lysis by effectors stimulated with the 15-mers, identified two overlapping HLA-B8- restricted CTL epitopes. Available data indicate that the sequence of one CTL epitope is conserved and the other is variant among P. falciparum isolates. Circulating activated CTL against the conserved epitope could be directly identified in one of the two volunteers. The identification of two HLA-B8-restricted CTL epitopes on PfSSP2 provides data critical to developing an epitope-based anti-liver stage malaria vaccine.
PMCID: PMC2192212  PMID: 7595214
23.  Single Immunization With a Monovalent Vesicular Stomatitis Virus–Based Vaccine Protects Nonhuman Primates Against Heterologous Challenge With Bundibugyo ebolavirus 
The Journal of Infectious Diseases  2011;204(Suppl 3):S1082-S1089.
The recombinant vesicular stomatitis virus (rVSV) vector-based monovalent vaccine platform expressing a filovirus glycoprotein has been demonstrated to provide protection from lethal challenge with Ebola (EBOV) and Marburg (MARV) viruses both prophylactically and after exposure. This platform provides protection between heterologous strains within a species; however, protection from lethal challenge between species has been largely unsuccessful. To determine whether the rVSV-EBOV vaccines have the potential to provide protection against a newly emerging, phylogenetically related species, cynomolgus macaques were vaccinated with an rVSV vaccine expressing either the glycoprotein of Zaire ebolavirus (ZEBOV) or Côte d’Ivoire ebolavirus (CIEBOV) and then challenged with Bundibugyo ebolavirus (BEBOV), which was recently proposed as a new EBOV species following an outbreak in Uganda in 2007. A single vaccination with the ZEBOV–specific vaccine provided cross-protection (75% survival) against subsequent BEBOV challenge, whereas vaccination with the CIEBOV–specific vaccine resulted in an outcome similar to mock-immunized animals (33% and 25% survival, respectively). This demonstrates that monovalent rVSV-based vaccines may be useful against a newly emerging species; however, heterologous protection across species remains challenging and may depend on enhancing the immune responses either through booster immunizations or through the inclusion of multiple immunogens.
doi:10.1093/infdis/jir350
PMCID: PMC3189995  PMID: 21987745
24.  Epitope-Based Vaccine Target Screening against Highly Pathogenic MERS-CoV: An In Silico Approach Applied to Emerging Infectious Diseases 
PLoS ONE  2015;10(12):e0144475.
Middle East respiratory syndrome coronavirus (MERS-CoV) with pandemic potential is a major worldwide threat to public health. However, vaccine development for this pathogen lags behind as immunity associated with protection is currently largely unknown. In this study, an immunoinformatics-driven genome-wide screening strategy of vaccine targets was performed to thoroughly screen the vital and effective dominant immunogens against MERS-CoV. Conservancy and population coverage analysis of the epitopes were done by the Immune Epitope Database. The results showed that the nucleocapsid (N) protein of MERS-CoV might be a better protective immunogen with high conservancy and potential eliciting both neutralizing antibodies and T-cell responses compared with spike (S) protein. Further, the B-cell, helper T-cell and cytotoxic T lymphocyte (CTL) epitopes were screened and mapped to the N protein. A total of 15 linear and 10 conformal B-cell epitopes that may induce protective neutralizing antibodies were obtained. Additionally, a total of 71 peptides with 9-mer core sequence were identified as helper T-cell epitopes, and 34 peptides were identified as CTL epitopes. Based on the maximum HLA binding alleles, top 10 helper T-cell epitopes and CTL epitopes that may elicit protective cellular immune responses against MERS-CoV were selected as MERS vaccine candidates. Population coverage analysis showed that the putative helper T-cell epitopes and CTL epitopes could cover the vast majority of the population in 15 geographic regions considered where vaccine would be employed. The B- and T-cell stimulation potentials of the screened epitopes is to be further validated for their efficient use as vaccines against MERS-CoV. Collectively, this study provides novel vaccine target candidates and may prompt further development of vaccines against MERS-CoV and other emerging infectious diseases.
doi:10.1371/journal.pone.0144475
PMCID: PMC4671582  PMID: 26641892
25.  Dynamics of Immune Escape during HIV/SIV Infection 
PLoS Computational Biology  2008;4(7):e1000103.
Several studies have shown that cytotoxic T lymphocytes (CTLs) play an important role in controlling HIV/SIV infection. Notably, the observation of escape mutants suggests a selective pressure induced by the CTL response. However, it remains difficult to assess the definite role of the cellular immune response. We devise a computational model of HIV/SIV infection having a broad cellular immune response targeting different viral epitopes. The CTL clones are stimulated by viral antigen and interact with the virus population through cytotoxic killing of infected cells. Consequently, the virus population reacts through the acquisition of CTL escape mutations. Our model provides realistic virus dynamics and describes several experimental observations. We postulate that inter-clonal competition and immunodominance may be critical factors determining the sequential emergence of escapes. We show that even though the total killing induced by the CTL response can be high, escape rates against a single CTL clone are often slow and difficult to estimate from infrequent sequence measurements. Finally, our simulations show that a higher degree of immunodominance leads to more frequent escape with a reduced control of viral replication but a substantially impaired replicative capacity of the virus. This result suggests two strategies for vaccine design: Vaccines inducing a broad CTL response should decrease the viral load, whereas vaccines stimulating a narrow but dominant CTL response are likely to induce escape but may dramatically reduce the replicative capacity of the virus.
Author Summary
As a result of their high mutation rate, HIV and its counterpart SIV in non-human primates can evade recognition by the host immune response through the generation of viral variants, the so-called escape mutants. This avoidance of cytotoxic T lymphocyte (CTL) mediated killing seems to be one of the major reasons why virus replication is not controlled effectively. However, it remains difficult to investigate the critical properties of the dynamics of immune escape. To this end, we developed a new computational model of HIV/SIV infection consisting of several CTL clones that can recognize specific parts of viral proteins, i.e., epitopes. The simulations allow us to follow the dynamics of immune escape in detail and help to interpret longitudinal data of HIV/SIV infections. Interestingly, changing the relative sizes of the CTL clones leads to a different evolution of the virus. Instead of reducing the number of infected cells, an alternative strategy of vaccine design could be to reduce the replicative capacity of the virus that might have implications for disease progression.
doi:10.1371/journal.pcbi.1000103
PMCID: PMC2423483  PMID: 18636096

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