Edward T. Mee

MHC Heterozygote Advantage in Simian Immunodeficiency Virus–Infected Mauritian Cynomolgus Macaques

This manuscript demonstrates unambiguous major histocompatibility complex heterozygote advantage in macaque monkeys infected with the same strain of simian immunodeficiency virus, suggesting that a prophylactic HIV vaccine should elicit a population of CD8+ T cells with broad specificity. A Broad View of HIV Some studies of HIV-infected people have suggested that HIV is better controlled when the individual’s immune response is broader, that is, when more parts of the HIV virus are recognized by T cells. Indeed, the lack of a broad immune response may explain why HIV vaccines have generally not been successful. Despite the importance of this question for vaccine design, it has been difficult to answer definitively because of diversity in HIV strain, sampling time after infection, individual genetics, and other variables. Now, O’Connor et al. use genetically defined Mauritian cynomolgus macaques to get around these issues and test whether a broader immune response does in fact lead to better disease control. The immune response to a virus is determined in part by the genetics at the HLA locus. This locus is important because variability in HLA class I genes determines the number of major histocompatibility complex (MHC) molecules generated; the number of MHC molecules then determines the number of epitopes that can be presented to immune CD8 T cells. Individuals who are heterozygotes at this locus are expected to have a broader immune response than do homozygotes because they have the potential to present a more diverse set of epitopes to immune cells. O’Connor and colleagues measured viral blood concentrations and cellular immune responses in cynomolgus macaques harboring identical MHC genetics and infected with the same strain of simian immunodeficiency virus; this enabled them to unambiguously define the relationship among MHC diversity, CD8 T cell breadth, and disease outcome. They found that the vast majority of macaques homozygous for MHC had viral loads nearly 80 times those of their heterozygote counterparts; the associated CD8 T cell responses, measured by immune assays that rely on visualization techniques, were inconsistent. Therefore, to better understand their results, the authors examined how the animals’ CD8 T cell epitopes changed with time. They found that viral sequences isolated from MHC heterozygotes collected 1 year after infection matched variants observed in each of their MHC homozygote counterparts at 1 year after infection, which suggested that the CD8 T cell responses in MHC heterozygotes were an assemblage of the responses from their MHC homozygote counterparts. These data collectively indicate that the potential breadth of the immune response determines viral replication: The broader the response, the less replication. This study builds on previous observational studies showing heterozygote advantage in HIV-infected people, and sets the stage for future studies exploring the mechanisms responsible for this immunological control of immunodeficiency viruses. Furthermore, through the use of these macaques with identical MHC genetics, vaccine candidates can be tested for their effectiveness in the presence of limited CD8 T lymphocyte diversity. The importance of a broad CD8 T lymphocyte (CD8-TL) immune response to HIV is unknown. Ex vivo measurements of immunological activity directed at a limited number of defined epitopes provide an incomplete portrait of the actual immune response. We examined viral loads in simian immunodeficiency virus (SIV)–infected major histocompatibility complex (MHC)–homozygous and MHC-heterozygous Mauritian cynomolgus macaques. Chronic viremia in MHC-homozygous macaques was 80 times that in MHC-heterozygous macaques. Virus from MHC-homozygous macaques accumulated 11 to 14 variants, consistent with escape from CD8-TL responses after 1 year of SIV infection. The pattern of mutations detected in MHC-heterozygous macaques suggests that their epitope-specific CD8-TL responses are a composite of those present in their MHC-homozygous counterparts. These results provide the clearest example of MHC heterozygote advantage among individuals infected with the same immunodeficiency virus strain, suggesting that broad recognition of multiple CD8-TL epitopes should be a key feature of HIV vaccines.

Mhc haplotype H6 is associated with sustained control of SIVmac251 infection in Mauritian cynomolgus macaques

The restricted diversity of the major histocompatibility complex (MHC) of Mauritian cynomolgus macaques provides powerful opportunities for insight into host-viral interactions and cellular immune responses that restrict lentiviral infections. However, little is known about the effects of Mhc haplotypes on control of SIV in this species. Using microsatellite-based genotyping and allele-specific PCR, Mhc haplotypes were deduced for 35 macaques infected with the same stock of SIVmac251. Class I haplotype H6 was associated with a reduction in chronic phase viraemia (p = 0.0145) while a similar association was observed for H6 class II (p = 0.0063). An increase in chronic phase viraemia, albeit an insignificant trend, was observed in haplotype H5-positive animals. These results further emphasise the value of genetically defined populations of non-human primates in AIDS research and provide a foundation for detailed characterisation of MHC restricted cellular immune responses and the effects of host genetics on SIV replication in cynomolgus macaques.

MHC haplotype frequencies in a UK breeding colony of Mauritian cynomolgus macaques mirror those found in a distinct population from the same geographic origin.

Background  Mauritian cynomolgus macaques have greatly restricted genetic diversity in the MHC region compared to other non‐human primates; however, the frequency of common MHC haplotypes among captive‐bred populations has not been reported.

Immunogenicity of a Recombinant Measles-HIV-1 Clade B Candidate Vaccine

Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 105 TCID50 of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.

Early Potent Protection against Heterologous SIVsmE660 Challenge Following Live Attenuated SIV Vaccination in Mauritian Cynomolgus Macaques

Background Live attenuated simian immunodeficiency virus (SIV) vaccines represent the most effective means of vaccinating macaques against pathogenic SIV challenge. However, thus far, protection has been demonstrated to be more effective against homologous than heterologous strains. Immune correlates of vaccine-induced protection have also been difficult to identify, particularly those measurable in the peripheral circulation. Methodology/Principal Findings Here we describe potent protection in 6 out of 8 Mauritian-derived cynomolgus macaques (MCM) against heterologous virus challenge with the pathogenic, uncloned SIVsmE660 viral stock following vaccination with live attenuated SIVmac251/C8. MCM provided a characterised host genetic background with limited Major Histocompatibility Complex (MHC) and TRIM5α allelic diversity. Early protection, observed as soon as 3 weeks post-vaccination, was comparable to that of 20 weeks vaccination. Recrudescence of vaccine virus was most pronounced in breakthrough cases where simultaneous identification of vaccine and challenge viruses by virus-specific PCR was indicative of active co-infection. Persistence of the vaccine virus in a range of lymphoid tissues was typified by a consistent level of SIV RNA positive cells in protected vaccinates. However, no association between MHC class I /II haplotype or TRIM5α polymorphism and study outcome was identified. Conclusion/Significance This SIV vaccine study, conducted in MHC-characterised MCM, demonstrated potent protection against the pathogenic, heterologous SIVsmE660 challenge stock after only 3 weeks vaccination. This level of protection against this viral stock by intravenous challenge has not been hitherto observed. The mechanism(s) of protection by vaccination with live attenuated SIV must account for the heterologous and early protection data described in this study, including those which relate to the innate immune system.

Mhc haplotype M3 is associated with early control of SHIVsbg infection in Mauritian cynomolgus macaques

The restricted major histocompatibilty complex of Mauritian cynomolgus macaques confers exceptional potential on this species in human immunodeficiency virus (HIV) vaccine development. However, knowledge of the effects of Mhc genetics on commonly used simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) stocks is incomplete. We determined the effect of Mhc haplotypes on SHIVsbg replication kinetics in a cohort of 25 naïve cynomolgus macaques. Haplotype M3 was associated with a 1.58log(10) reduction in viraemia at day 28 post infection (p.i.). Haplotype M6 was associated with elevated SHIVsbg viraemia at days 28 and 56. No significant effect of Mhc class II haplotypes on viral replication was observed. These data emphasise the importance of genetic characterisation of experimental macaques and advance our understanding of host genetic effects in SIV/SHIV models of HIV infection.

Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing

Abstract Background Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity. Methods A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay. Results Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4–14 laboratories. Six non-target viruses were detected by three or more laboratories. Conclusion The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories.

Immunogenicity of a recombinant measles HIV-1 subtype C vaccine.

The HIV epidemic is greatest in Sub-Saharan Africa and India where HIV-1 subtype C is predominant. To control the spread of HIV in these parts of the world a preventive HIV-1 subtype C vaccine is urgently required. Here we report the immunogenicity of a candidate HIV-1 subtype C vaccine delivered by a recombinant measles vector carrying an insert encoding HIV-1 subtype C Gag, RT and Nef (MV1-F4), in MHC-typed non-human primates. HIV-1 specific cytokine secreting CD4+ and CD8+ T cell responses were detected in 15 out of 16 vaccinees. These HIV-specific T cell responses persisted in lymphoid tissues. Anti-HIV-1 antibody responses were detected in 15 out of 16 vaccinees and titres were boosted by a second immunisation carried out 84 days later. These findings support further exploration of the MV1-F4 vector as a candidate HIV-1 subtype C vaccine or as part of a wider vaccine strategy.

Challenges of the Unknown: Clinical Application of Microbial Metagenomics

Availability of fast, high throughput and low cost whole genome sequencing holds great promise within public health microbiology, with applications ranging from outbreak detection and tracking transmission events to understanding the role played by microbial communities in health and disease. Within clinical metagenomics, identifying microorganisms from a complex and host enriched background remains a central computational challenge. As proof of principle, we sequenced two metagenomic samples, a known viral mixture of 25 human pathogens and an unknown complex biological model using benchtop technology. The datasets were then analysed using a bioinformatic pipeline developed around recent fast classification methods. A targeted approach was able to detect 20 of the viruses against a background of host contamination from multiple sources and bacterial contamination. An alternative untargeted identification method was highly correlated with these classifications, and over 1,600 species were identified when applied to the complex biological model, including several species captured at over 50% genome coverage. In summary, this study demonstrates the great potential of applying metagenomics within the clinical laboratory setting and that this can be achieved using infrastructure available to nondedicated sequencing centres.

High resolution identity testing of inactivated poliovirus vaccines

Highlights • Identity testing is a critical step in the quality control process.• Serological testing is the current approved method, but has certain limitations.• Existing molecular methods (qPCR) provide information about small genomic regions.• Random amplification and shotgun sequencing provide full genome coverage.• Distinction of highly similar viruses, and manufacturer-specific differences is possible.