The live attenuated simian immunodeficiency virus (LASIV) vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design.

AIDS Research and Human RetrovirusesVol. 30, No. S1 ImmunogensFree AccessCD8 T-cell Based HIV Vaccines - Is Targeting Conserved Epitopes the Answer?Shelby L. O'Connor, Dane Gellerup, Max Harris, and Ericka BeckerShelby L. O'ConnorSearch for more papers by this author, Dane GellerupSearch for more papers by this author, Max HarrisSearch for more papers by this author, and Ericka BeckerSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5226.abstractAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail P10.06Background: Immunodominant CD8 T cell responses emerge in the first few weeks after HIV/SIV infection, suppress virus replication, and select for escape variants. Although T-cell based HIV vaccines have not successfully provided sterilizing immunity, vaccine-elicited CD8 T cells may contribute to the control of replication of breakthrough viruses. It is critical that a T-cell based vaccine generates an effective pool of memory CD8 T cells that are available to respond during acute HIV infection and effectively control both acute and chronic virus replication. Designing a vaccine to elicit these potent CD8 T cells in all vaccinated individuals, however, is a daunting challenge. Conceptually, a vaccine that elicits CD8 T cell responses targeting highly conserved virus peptide sequences would have the most widespread impact, but the efficacy of T cells targeting these conserved epitopes is unknown.Methods: We employ a model of SIVmac239Δnef-infected MHC-identical Mauritian cynomolgus macaques to test the hypothesis that CD8 T cells targeting conserved epitopes are unable to detect and destroy virally infected cells. Accordingly, we expect that CD8 T cells targeting epitopes that accumulate variants are more effective at controlling virus replication. To test this hypothesis, we are creating variants of live attenuated SIVmac239Δnef designed to elicit CD8 T cells targeting epitopes that do and do not accumulate variants.Results: We will determine whether the mutant viruses are ‘fit’ and whether the included variant epitope sequences are no longer detected by CD8 T cells. In the future, we will determine if acute CD8 T cells targeting these different categories of epitopes are able to control virus replication, in vivo.Conclusions: The conclusions from this study will help identify whether CD8 T cells that develop during acute HIV infection can be specific for highly conserved epitopes and whether they can control virus replication.FiguresReferencesRelatedDetails Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Shelby L. O'Connor, Dane Gellerup, Max Harris, and Ericka Becker.CD8 T-cell Based HIV Vaccines - Is Targeting Conserved Epitopes the Answer?.AIDS Research and Human Retroviruses.Oct 2014.A119-A119.http://doi.org/10.1089/aid.2014.5226.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

The overall CD8 T cell response to human/simian immunodeficiency virus (HIV/SIV) targets a collection of discrete epitope specificities. Some of these epitope-specific CD8 T cells emerge in the weeks and months following infection and rapidly select for sequence variants, whereas other CD8 T cell responses develop during the chronic infection phase and rarely select for sequence variants. In this study, we tested the hypothesis that acute-phase CD8 T cell responses that do not rapidly select for escape variants are unable to control viral replication in vivo as well as those that do rapidly select for escape variants. We created a derivative of live attenuated SIV (SIVmac239Δnef) in which we ablated five epitopes that elicit early CD8 T cell responses and rapidly accumulate sequence variants in SIVmac239-infected Mauritian cynomolgus macaques (MCMs) that are homozygous for the M3 major histocompatibility complex (MHC) haplotype. This live attenuated SIV variant was called m3KOΔnef. Viremia was significantly higher in M3 homozygous MCMs infected with m3KOΔnef than in either MHC-mismatched MCMs infected with m3KOΔnef or MCMs infected with SIVmac239Δnef. Three CD8 T cell responses, including two that do not rapidly select for escape variants, predominated during early m3KOΔnef infection in the M3 homozygous MCMs, but these animals were unable to control viral replication. These results provide evidence that acute-phase CD8 T cell responses that have the potential to rapidly select for escape variants in the early phase of infection are needed to establish viral control in vivo.

Specific major histocompatibility complex (MHC) class I alleles are associated with an increased frequency of spontaneous con-trol of human and simian immunodeficiency viruses (HIV and SIV). The mechanism of control is thought to involve MHC class I-restricted CD8 (cid:1) T cells, but it is not clear whether particular CD8 (cid:1) T cell responses or a broad repertoire of epitope-specific CD8 (cid:1) 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-homozy-gous and MHC-heterozygous cynomolgus macaques with the pathogenic virus SIVmac239. As measured by a gamma interferon enzyme-linked immunosorbent

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 (Gag 386-394 GW9, Nef 103-111 RM9, and Rev 59-68 SP10) targeted by immunodominant CD8 + T cell responses in acutely infected Mauritian cynomolgus macaques. In animals recognizing all three epitopes, variation within Rev 59-68 SP10 was associated with delayed accumulation of variants in Gag 386-394 GW9 but had no effect on variation within Nef 103-111 RM9. 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.

Deep sequencing technology is revolutionizing our understanding of HIV/SIV evolution. It is known that acute SIV sequence variation within CD8 T lymphocyte (CD8-TL) epitopes is similar among MHC-identical animals, but we do not know whether this persists into the chronic phase. We now determine whether chronic viral variation in MHC-identical animals infected with clonal SIV is similar throughout the entire coding sequence when using a sensitive deep sequencing approach. We pyrosequenced the entire coding sequence of the SIV genome isolated from a unique cohort of four SIVmac239-infected, MHC-identical Mauritian cynomolgus macaques (MCM) 48 weeks after infection; one MCM in the cohort became an elite controller. Among the three non-controllers, we found that genome-wide sequences were similar between animals and we detected increased sequence complexity within 64% of CD8-TL epitopes when compared to Sanger sequencing methods. When we compared sequences between the MHC-matched controller and the three non-controllers, we found the viral population in the controller was less diverse and accumulated different variants than the viral populations in the non-controllers. Importantly, we found that initial PCR amplification of viral cDNA did not significantly affect the sequences detected, suggesting that data obtained by pyrosequencing PCR-amplified viral cDNA accurately represents the diversity of sequences replicating within an animal. This demonstrates that chronic sequence diversity across the entire SIV coding sequence is similar among MHC-identical animals with comparable viral loads when infected with the same clonal virus stock. Additionally, our approach to genome-wide SIV sequencing accurately reflects the diversity of sequences present in the replicating viral population. In sum, our study suggests that genome-wide pyrosequencing of immunodeficiency viruses captures a thorough and unbiased picture of sequence diversity, and may be a useful approach to employ when evaluating which sequences to include as part of a vaccine immunogen.

Published Ahead of Print 9 May 2012. 2012, 86(14):7596. DOI: 10.1128/JVI.00716-12. J. Virol. O'Connor Gostick, David A. Price, Thomas C. Friedrich and David H. O'Connor, Michael Piatak Jr., Jeffrey D. Lifson, Emma Ericka A. Becker, Max Harris, Jason T. Weinfurter, Shelby L. Ericsen, Matthew Scarlotta, Brian T. Cain, Ngoc H. Pham, Melisa L. Budde, Justin M. Greene, Emily N. Chin, Adam J. Macaques Virus Replication in Mauritian Cynomolgus with Control of Simian Immunodeficiency T Cell Responses Correlate + Specific CD8

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.