Simon M. Lank

Biological and structural characterization of a host-adapting amino acid in influenza virus.

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the proteins interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals.

Establishment of Human Papillomavirus Infection Requires Cell Cycle Progression

Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis) for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these results also have potentially important implications for HPV control.

Major histocompatibility complex genotyping with massively parallel pyrosequencing

Major histocompatibility complex (MHC) genetics dictate adaptive cellular immune responses, making robust MHC genotyping methods essential for studies of infectious disease, vaccine development and transplantation. Nonhuman primates provide essential preclinical models for these areas of biomedical research. Unfortunately, given the unparalleled complexity of macaque MHCs, existing methodologies are inadequate for MHC typing of these key model animals. Here we use pyrosequencing of complementary DNA–PCR amplicons as a general approach to determine comprehensive MHC class I genotypes in nonhuman primates. More than 500 unique MHC class I sequences were resolved by sequence-based typing of rhesus, cynomolgus and pig-tailed macaques, nearly half of which have not been reported previously. The remarkable sensitivity of this approach in macaques demonstrates that pyrosequencing is viable for ultra–high-throughput MHC genotyping of primates, including humans.

Ultradeep Pyrosequencing Detects Complex Patterns of CD8+ T-Lymphocyte Escape in Simian Immunodeficiency Virus-Infected Macaques

ABSTRACT Human and simian immunodeficiency viruses (HIV/SIV) exhibit enormous sequence heterogeneity within each infected host. Here, we use ultradeep pyrosequencing to create a comprehensive picture of CD8+ T-lymphocyte (CD8-TL) escape in SIV-infected macaques, revealing a previously undetected complex pattern of viral variants. This increased sensitivity enabled the detection of acute CD8-TL escape as early as 17 days postinfection, representing the earliest published example of CD8-TL escape in intrarectally infected macaques. These data demonstrate that pyrosequencing can be used to study the evolution of CD8-TL escape during immunodeficiency virus infection with an unprecedented degree of sensitivity.

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.

Novel, Divergent Simian Hemorrhagic Fever Viruses in a Wild Ugandan Red Colobus Monkey Discovered Using Direct Pyrosequencing

Background Simian hemorrhagic fever virus (SHFV) has caused lethal outbreaks of hemorrhagic disease in captive primates, but its distribution in wild primates has remained obscure. Here, we describe the discovery and genetic characterization by direct pyrosequencing of two novel, divergent SHFV variants co-infecting a single male red colobus monkey from Kibale National Park, Uganda. Methodology/Principal Findings The viruses were detected directly from blood plasma using pyrosequencing, without prior virus isolation and with minimal PCR amplification. The two new SHFV variants, SHFV-krc1 and SHFV-krc2 are highly divergent from each other (51.9% nucleotide sequence identity) and from the SHFV type strain LVR 42-0/M6941 (52.0% and 51.8% nucleotide sequence identity, respectively) and demonstrate greater phylogenetic diversity within SHFV than has been documented within any other arterivirus. Both new variants nevertheless have the same 3′ genomic architecture as the type strain, containing three open reading frames not present in the other arteriviruses. Conclusions/Significance These results represent the first documentation of SHFV in a wild primate and confirm the unusual 3′ genetic architecture of SHFV relative to the other arteriviruses. They also demonstrate a degree of evolutionary divergence within SHFV that is roughly equivalent to the degree of divergence between other arterivirus species. The presence of two such highly divergent SHFV variants co-infecting a single individual represents a degree of within-host viral diversity that exceeds what has previously been reported for any arterivirus. These results expand our knowledge of the natural history and diversity of the arteriviruses and underscore the importance of wild primates as reservoirs for novel pathogens.

A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping

Human leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans ambiguities, hindering both studies correlating high-resolution genotype with clinical outcomes, and population-specific allele frequency surveys. We present an assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence-based typing. The amplicon is predicted to unambiguously resolve 85% of known alleles. A panel of 48 previously HLA-typed samples was assayed with this method, demonstrating 100% non-null allele typing concordance. We show that this technique can multiplex at least 768 patients per sequencing run with multiplex identifier sequence bar-coding. Unprecedented typing throughput results from a novel single cDNA-PCR amplicon strategy requiring only 1 PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.

Infection with “Escaped” Virus Variants Impairs Control of Simian Immunodeficiency Virus SIVmac239 Replication in Mamu-B*08-Positive Macaques

ABSTRACT An understanding of the mechanism(s) by which some individuals spontaneously control human immunodeficiency virus (HIV)/simian immunodeficiency virus replication may aid vaccine design. Approximately 50% of Indian rhesus macaques that express the major histocompatibility complex (MHC) class I allele Mamu-B*08 become elite controllers after infection with simian immunodeficiency virus SIVmac239. Mamu-B*08 has a binding motif that is very similar to that of HLA-B27, a human MHC class I allele associated with the elite control of HIV, suggesting that SIVmac239-infected Mamu-B*08-positive (Mamu-B*08+) animals may be a good model for the elite control of HIV. The association with MHC class I alleles implicates CD8+ T cells and/or natural killer cells in the control of viral replication. We therefore introduced point mutations into eight Mamu-B*08-restricted CD8+ T-cell epitopes to investigate the contribution of epitope-specific CD8+ T-cell responses to the development of the control of viral replication. Ten Mamu-B*08+ macaques were infected with this mutant virus, 8X-SIVmac239. We compared immune responses and viral loads of these animals to those of wild-type SIVmac239-infected Mamu-B*08+ macaques. The five most immunodominant Mamu-B*08-restricted CD8+ T-cell responses were barely detectable in 8X-SIVmac239-infected animals. By 48 weeks postinfection, 2 of 10 8X-SIVmac239-infected Mamu-B*08+ animals controlled viral replication to <20,000 viral RNA (vRNA) copy equivalents (eq)/ml plasma, while 10 of 15 wild-type-infected Mamu-B*08+ animals had viral loads of <20,000 vRNA copy eq/ml (P = 0.04). Our results suggest that these epitope-specific CD8+ T-cell responses may play a role in establishing the control of viral replication in Mamu-B*08+ macaques.

Identification of novel MHC class I sequences in pig-tailed macaques by amplicon pyrosequencing and full-length cDNA cloning and sequencing

Pig-tailed macaques (Macaca nemestrina) provide important animal models in biomedical research, but utility of this species for HIV and other disease pathogenesis research is limited by incomplete knowledge of major histocompatibility complex (MHC) class I genetics. Here, we describe comprehensive MHC class I genotyping of 24 pig-tailed macaques, using pyrosequencing to evaluate a 367- bp complementary DNA (cDNA)-PCR amplicon spanning the highly polymorphic peptide-binding region of MHC class I transcripts. We detected 29 previously described Mane transcripts, 90 novel class I sequences, and eight shared MHC class IB haplotypes. We used this genotyping data to inform full-length MHC class I cDNA allele discovery, characterizing 66 novel full-length transcripts. These new full-length sequences nearly triple the number of Mane-B cDNA sequences previously characterized. The comprehensive genotypes and full-length Mane transcripts described herein add value to pig-tailed macaques as model organisms in biomedical research; furthermore, the coordinated method for MHC genotyping and allele discovery is extensible to other less well-characterized nonhuman primate species.