Ericka A. Becker

Comprehensive characterization of MHC class II haplotypes in Mauritian cynomolgus macaques

There are currently no nonhuman primate models with fully defined major histocompatibility complex (MHC) class II genetics. We recently showed that six common MHC haplotypes account for essentially all MHC diversity in cynomolgus macaques (Macaca fascicularis) from the island of Mauritius. In this study, we employ complementary DNA cloning and sequencing to comprehensively characterize full length MHC class II alleles expressed at the Mafa-DPA, -DPB, -DQA, -DQB, -DRA, and -DRB loci on the six common haplotypes. We describe 34 full-length MHC class II alleles, 12 of which are completely novel. Polymorphism was evident at all six loci including DPA, a locus thought to be monomorphic in rhesus macaques. Similar to other Old World monkeys, Mauritian cynomolgus macaques (MCM) share MHC class II allelic lineages with humans at the DQ and DR loci, but not at the DP loci. Additionally, we identified extensive sharing of MHC class II alleles between MCM and other nonhuman primates. The characterization of these full-length-expressed MHC class II alleles will enable researchers to generate MHC class II transferent cell lines, tetramers, and other molecular reagents that can be used to explore CD4+ T lymphocyte responses in MCM.

Whole-genome characterization of human and simian immunodeficiency virus intrahost diversity by ultradeep pyrosequencing.

ABSTRACT Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.

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.

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

ABSTRACT Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naïve patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

MHC class I characterization of Indonesian cynomolgus macaques

Cynomolgus macaques (Macaca fascicularis) are quickly becoming a useful model for infectious disease and transplantation research. Even though cynomolgus macaques from different geographic regions are used for these studies, there has been limited characterization of full-length major histocompatibility complex (MHC) class I immunogenetics of distinct geographic populations. Here, we identified 48 MHC class I cDNA nucleotide sequences in eleven Indonesian cynomolgus macaques, including 41 novel Mafa-A and Mafa-B sequences. We found seven MHC class I sequences in Indonesian macaques that were identical to MHC class I sequences identified in Malaysian or Mauritian macaques. Sharing of nucleotide sequences between these geographically distinct populations is also consistent with the hypothesis that Indonesia was a source of the Mauritian macaque population. In addition, we found that the Indonesian cDNA sequence Mafa-B*7601 is identical throughout its peptide binding domain to Mamu-B*03, an allele that has been associated with control of Simian immunodeficiency virus (SIV) viremia in Indian rhesus macaques. Overall, a better understanding of the MHC class I alleles present in Indonesian cynomolgus macaques improves their value as a model for disease research, and it better defines the biogeography of cynomolgus macaques throughout Southeast Asia.

Transcriptionally Abundant Major Histocompatibility Complex Class I Alleles Are Fundamental to Nonhuman Primate Simian Immunodeficiency Virus-Specific CD8+ T Cell Responses

ABSTRACT Simian immunodeficiency virus (SIV)-infected macaques are the preferred animal model for human immunodeficiency virus (HIV) vaccines that elicit CD8+ T cell responses. Unlike humans, whose CD8+ T cell responses are restricted by a maximum of six HLA class I alleles, macaques express up to 20 distinct major histocompatibility complex class I (MHC-I) sequences. Interestingly, only a subset of macaque MHC-I sequences are transcriptionally abundant in peripheral blood lymphocytes. We hypothesized that highly transcribed MHC-I sequences are principally responsible for restricting SIV-specific CD8+ T cell responses. To examine this hypothesis, we measured SIV-specific CD8+ T cell responses in MHC-I homozygous Mauritian cynomolgus macaques. Each of eight CD8+ T cell responses defined by full-proteome gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay were restricted by four of the five transcripts that are transcriptionally abundant (>1% of total MHC-I transcripts in peripheral blood lymphocytes). The five transcriptionally rare transcripts shared by these animals did not restrict any detectable CD8+ T cell responses. Further, seven CD8+ T cell responses were defined by identifying peptide binding motifs of the three most frequent MHC-I transcripts on the M3 haplotype. Combined, these results suggest that transcriptionally abundant MHC-I transcripts are principally responsible for restricting SIV-specific CD8+ T cell responses. Thus, only a subset of the thousands of known MHC-I alleles in macaques should be prioritized for CD8+ T cell epitope characterization.

Specific CD8+ T Cell Responses Correlate with Control of Simian Immunodeficiency Virus Replication in Mauritian Cynomolgus Macaques

ABSTRACT Specific major histocompatibility complex (MHC) class I alleles are associated with an increased frequency of spontaneous control of human and simian immunodeficiency viruses (HIV and SIV). The mechanism of control is thought to involve MHC class I-restricted CD8+ T cells, but it is not clear whether particular CD8+ T cell responses or a broad repertoire of epitope-specific CD8+ T cell populations (termed T cell breadth) are principally responsible for mediating immunologic control. To test the hypothesis that heterozygous macaques control SIV replication as a function of superior T cell breadth, we infected MHC-homozygous and MHC-heterozygous cynomolgus macaques with the pathogenic virus SIVmac239. As measured by a gamma interferon enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT) using blood, T cell breadth did not differ significantly between homozygotes and heterozygotes. Surprisingly, macaques that controlled SIV replication, regardless of their MHC zygosity, shared durable T cell responses against similar regions of Nef. While the limited genetic variability in these animals prevents us from making generalizations about the importance of Nef-specific T cell responses in controlling HIV, these results suggest that the T cell-mediated control of virus replication that we observed is more likely the consequence of targeting specificity rather than T cell breadth.

Conditional CD8+ T Cell Escape during Acute Simian Immunodeficiency Virus Infection

ABSTRACT CD8+ T cell responses rapidly select viral variants during acute human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. We used pyrosequencing to examine variation within three SIV-derived epitopes (Gag386-394GW9, Nef103-111RM9, and Rev59-68SP10) targeted by immunodominant CD8+ T cell responses in acutely infected Mauritian cynomolgus macaques. In animals recognizing all three epitopes, variation within Rev59-68SP10 was associated with delayed accumulation of variants in Gag386-394GW9 but had no effect on variation within Nef103-111RM9. This demonstrates that the entire T cell repertoire, rather than a single T cell population, influences the timing of immune escape, thereby providing the first example of conditional CD8+ T cell escape in HIV/SIV infection.

Characterization of full-length MHC class II sequences in Indonesian and Vietnamese cynomolgus macaques

In recent years, the use of cynomolgus macaques in biomedical research has increased greatly. However, with the exception of the Mauritian population, knowledge of the MHC class II genetics of the species remains limited. Here, using cDNA cloning and Sanger sequencing, we identified 127 full-length MHC class II alleles in a group of 12 Indonesian and 12 Vietnamese cynomolgus macaques. Forty two of these were completely novel to cynomolgus macaques while 61 extended the sequence of previously identified alleles from partial to full length. This more than doubles the number of full-length cynomolgus macaque MHC class II alleles available in GenBank, significantly expanding the allele library for the species and laying the groundwork for future evolutionary and functional studies.

Experimental Analysis of Sources of Error in Evolutionary Studies Based on Roche/454 Pyrosequencing of Viral Genomes

Factors affecting the reliability of Roche/454 pyrosequencing for analyzing sequence polymorphism in within-host viral populations were assessed by two experiments: 1) sequencing four clonal simian immunodeficiency virus (SIV) stocks and 2) sequencing mixtures in different proportions of two SIV strains with known fixed nucleotide differences. Observed nucleotide diversity and frequency of undetermined nucleotides were increased at sites in homopolymer runs of four or more identical nucleotides, particularly at AT sites. However, in the mixed-strain experiments, the effects on estimated nucleotide diversity of such errors were small in comparison to known strain differences. The results suggest that biologically meaningful variants present at a frequency of around 10% and possibly much lower are easily distinguished from artifacts of the sequencing process. Analysis of the clonal stocks revealed numerous rare variants that showed the signature of purifying selection and that elimination of variants at frequencies of less than 1% reduced estimates of nucleotide diversity by about an order of magnitude. Thus, using a 1% frequency cutoff for accepting a variant as real represents a conservative standard, which may be useful in studies that are focused on the discovery of specific mutations (such as those conferring immune escape or drug resistance). On the other hand, if the goal is to estimate nucleotide diversity, an optimal strategy might be to include all observed variants (even those at less than 1% frequency), while masking out homopolymer runs of four or more nucleotides.