john paul mcnevin

Publications

• 5.15

  Impact points

  Vpu mediates IRF3 depletion during HIV infection by a lysosomal-dependent mechanism.

  Brian P Doehle, Kristina Chang, Arjun Rustagi, John McNevin, M Juliana McElrath, Michael Gale

  Journal of virology. 05/2012;

  HIV has evolved sophisticated mechanisms to avoid restriction by intracellular innate immune defenses that otherwise serve to control acute viral infection and virus dissemination. Innate defenses are triggered when pathogen recognition receptor (PRR) proteins of the host cell engage pathogen-associ... [more] HIV has evolved sophisticated mechanisms to avoid restriction by intracellular innate immune defenses that otherwise serve to control acute viral infection and virus dissemination. Innate defenses are triggered when pathogen recognition receptor (PRR) proteins of the host cell engage pathogen-associated molecule patterns (PAMPs) present in viral products. Interferon regulatory factor (IRF)3 plays a central role in PRR signaling of innate immunity to drive the expression of type I interferon (IFN) and interferon-stimulated genes (ISGs), including a variety of HIV restriction factors, that serve to limit viral replication directly and/or program adaptive immunity. Productive infection of T cells by HIV is dependent upon the targeted proteolysis of IRF3 that occurs through a virus-directed mechanism that results in suppression of innate immune defenses. However, the mechanisms by which HIV controls innate immune signaling and IRF3 function are not defined. Here, we examined the innate immune response induced by HIV strains identified through their differential control of PRR signaling. We identified viruses that unlike typical circulating HIV strains lack the ability to degrade IRF3. Our studies show that IRF3 regulation maps specifically to the HIV accessory protein Vpu. We define a molecular interaction between Vpu and IRF3 that redirects IRF3 to the endo-lysosome for proteolytic degradation, thus allowing HIV to avoid the innate antiviral immune response. Our studies reveal Vpu as an important IRF3 regulator that supports acute HIV infection through innate immune suppression. These observations define the Vpu/IRF3 interface as a novel target for therapeutic strategies aimed at enhancing the immune response to HIV.
• 5.15

  Impact points

  Demographic processes affect HIV-1 evolution in primary infection before the onset of selective processes.

  Joshua T Herbeck, Morgane Rolland, Yi Liu, Sherry McLaughlin, John McNevin, Hong Zhao, Kim Wong, Julia N Stoddard, Dana Raugi, Stephanie Sorensen, Indira Genowati, Brian Birditt, Angela McKay, Kurt Diem, Brandon S Maust, Wenjie Deng, Ann C Collier, Joanne D Stekler, M Juliana McElrath, James I Mullins

  Journal of virology. 05/2011; 85(15):7523-34.

  HIV-1 transmission and viral evolution in the first year of infection were studied in 11 individuals representing four transmitter-recipient pairs and three independent seroconverters. Nine of these individuals were enrolled during acute infection; all were men who have sex with men (MSM) infected w... [more] HIV-1 transmission and viral evolution in the first year of infection were studied in 11 individuals representing four transmitter-recipient pairs and three independent seroconverters. Nine of these individuals were enrolled during acute infection; all were men who have sex with men (MSM) infected with HIV-1 subtype B. A total of 475 nearly full-length HIV-1 genome sequences were generated, representing on average 10 genomes per specimen at 2 to 12 visits over the first year of infection. Single founding variants with nearly homogeneous viral populations were detected in eight of the nine individuals who were enrolled during acute HIV-1 infection. Restriction to a single founder variant was not due to a lack of diversity in the transmitter as homogeneous populations were found in recipients from transmitters with chronic infection. Mutational patterns indicative of rapid viral population growth dominated during the first 5 weeks of infection and included a slight contraction of viral genetic diversity over the first 20 to 40 days. Subsequently, selection dominated, most markedly in env and nef. Mutants were detected in the first week and became consensus as early as day 21 after the onset of symptoms of primary HIV infection. We found multiple indications of cytotoxic T lymphocyte (CTL) escape mutations while reversions appeared limited. Putative escape mutations were often rapidly replaced with mutually exclusive mutations nearby, indicating the existence of a maturational escape process, possibly in adaptation to viral fitness constraints or to immune responses against new variants. We showed that establishment of HIV-1 infection is likely due to a biological mechanism that restricts transmission rather than to early adaptive evolution during acute infection. Furthermore, the diversity of HIV strains coupled with complex and individual-specific patterns of CTL escape did not reveal shared sequence characteristics of acute infection that could be harnessed for vaccine design.
• 4.41

  Impact points

  Dynamics of viral evolution and CTL responses in HIV-1 infection.

  Yi Liu, John P McNevin, Sarah Holte, M Juliana McElrath, James I Mullins

  PloS one. 01/2011; 6(1):e15639.

  Improved understanding of the dynamics of host immune responses and viral evolution is critical for effective HIV-1 vaccine design. We comprehensively analyzed Cytotoxic T-lymphocyte (CTL)-viral epitope dynamics in an antiretroviral therapy-naïve subject over the first four years of HIV-1 infection.... [more] Improved understanding of the dynamics of host immune responses and viral evolution is critical for effective HIV-1 vaccine design. We comprehensively analyzed Cytotoxic T-lymphocyte (CTL)-viral epitope dynamics in an antiretroviral therapy-naïve subject over the first four years of HIV-1 infection. We found that CTL responses developed sequentially and required constant antigenic stimulation for maintenance. CTL responses exerting strong selective pressure emerged early and led to rapid escape, proliferated rapidly and were predominant during acute/early infection. Although CTL responses to a few persistent epitopes developed over the first two months of infection, they proliferated slowly. As CTL epitopes were replaced by mutational variants, the corresponding responses immediately declined, most rapidly in the cases of strongly selected epitopes. CTL recognition of epitope variants, via cross-reactivity and de novo responses, was common throughout the period of study. Our data demonstrate that HIV-specific CTL responses, especially in the critical acute/early stage, were focused on regions that are prone to escape. Failure of CTL responses to strongly target functional or structurally critical regions of the virus, as well as the sequential cascade of CTL responses, followed closely by viral escape and decline of the corresponding responses, likely contribute to a lack of sustainable viral suppression. Focusing early and rapidly proliferating CTL on persistent epitopes may be essential for durable viral control in HIV-1 infection.

• Dynamics of Viral Evolution and CTL Responses in HIV-1 Infection

  Liu Y, McNevin JP, Holte S, McElrath MJ, Mullins JI

  PLoS One. 01/2011; 6(1).

  Improved understanding of the dynamics of host immune responses and viral evolution is critical for effective HIV-1 vaccine design. We comprehensively analyzed Cytotoxic T-lymphocyte (CTL)-viral epitope dynamics in an antiretroviral therapy-naïve subject over the first four years of HIV-1 infection.... [more] Improved understanding of the dynamics of host immune responses and viral evolution is critical for effective HIV-1 vaccine design. We comprehensively analyzed Cytotoxic T-lymphocyte (CTL)-viral epitope dynamics in an antiretroviral therapy-naïve subject over the first four years of HIV-1 infection. We found that CTL responses developed sequentially and required constant antigenic stimulation for maintenance. CTL responses exerting strong selective pressure emerged early and led to rapid escape, proliferated rapidly and were predominant during acute/early infection. Although CTL responses to a few persistent epitopes developed over the first two months of infection, they proliferated slowly. As CTL epitopes were replaced by mutational variants, the corresponding responses immediately declined, most rapidly in the cases of strongly selected epitopes. CTL recognition of epitope variants, via cross-reactivity and de novo responses, was common throughout the period of study. Our data demonstrate that HIV-specific CTL responses, especially in the critical acute/early stage, were focused on regions that are prone to escape. Failure of CTL responses to strongly target functional or structurally critical regions of the virus, as well as the sequential cascade of CTL responses, followed closely by viral escape and decline of the corresponding responses, likely contribute to a lack of sustainable viral suppression. Focusing early and rapidly proliferating CTL on persistent epitopes may be essential for durable viral control in HIV-1 infection
• 5.33

  Impact points

  Innate immune signaling induces high levels of TC-specific deaminase activity in primary monocyte-derived cells through expression of APOBEC3A isoforms.

  Beth K Thielen, John P McNevin, M Juliana McElrath, Brook Vander Stoep Hunt, Kevin C Klein, Jaisri R Lingappa

  The Journal of biological chemistry. 09/2010; 285(36):27753-66.

  In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (oth... [more] In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date. In contrast, here we report high levels of deaminase activity at TC motifs when whole PBMCs or isolated primary monocyte-derived cells were treated with interferon-alpha (IFNalpha) or IFNalpha-inducing toll-like receptor ligands. Induction of TC-specific deaminase activity required new transcription and translation and correlated with the appearance of two APOBEC3A (A3A) isoforms. Knockdown of A3A in monocytes with siRNA abolished TC-specific deaminase activity, confirming that A3A isoforms are responsible for all TC-specific deaminase activity observed. Both A3A isoforms appear to be enzymatically active; moreover, our mutational studies raise the possibility that the smaller isoform results from internal translational initiation. In contrast to the high levels of TC-specific activity observed in IFNalpha-treated monocytes, CC-specific activity remained low in PBMCs, suggesting that A3G deaminase activity is relatively inhibited, unlike that of A3A. Together, these findings suggest that deaminase activity of A3A isoforms in monocytes and macrophages may play an important role in host defense against viruses.
• 5.87

  Impact points

  Conserved HIV-1 Epitopes Continuously Elicit Subdominant Cytotoxic T-Lymphocyte Responses.

  Yi Liu, John McNevin, Morgane Rolland, Hong Zhao, Wenjie Deng, Janine Maenza, Claire E Stevens, Ann C Collier, M Juliana McElrath, James I Mullins

  The Journal of infectious diseases. 10/2009;

  Background. The epitope specificities and antiviral activities of class I HLA-restricted CD8(+) T cells, especially those induced during human immunodeficiency virus type 1 (HIV-1) primary infection, are important considerations in designing HIV-1 vaccines. Conserved epitopes may be more commonly an... [more] Background. The epitope specificities and antiviral activities of class I HLA-restricted CD8(+) T cells, especially those induced during human immunodeficiency virus type 1 (HIV-1) primary infection, are important considerations in designing HIV-1 vaccines. Conserved epitopes may be more commonly and persistently recognized than variable epitopes, as they may be more likely to be present in infecting viruses. However, some studies have shown preferential or similar targeting of variable versus conserved epitopes during primary infection. Methods. We analyzed cytotoxic T-lymphocyte (CTL) responses toward predefined conserved and variable epitopes in 45 subjects during primary ([Formula: see text]) and/or chronic infection ([Formula: see text]). Results. Conserved and variable CTL epitopes were recognized with similar probabilities, whereas conserved epitopes generally elicited subdominant responses during both primary and chronic infection. During primary infection, CTL responses against Gag versus responses against Env and variable epitopes tended to be associated with lower and higher viral loads, respectively. During chronic infection, Env-specific responses tended to be associated with lower CD4(+) cell counts. Conclusions. Subdominant CTL recognition of conserved HIV-1 epitopes commonly occurs from the primary through chronic stages of HIV-1 infection. These findings underscore the challenge in designing T cell-based vaccines that can induce immunodominant CTL responses to conserved HIV-1 regions.
• 5.15

  Impact points

  Pre-infection HIV-specific CTL failed to prevent HIV-1 infection from strains genetically unrelated to viruses in long-term exposed partner.

  Yi Liu, Amanda Woodward, Haiying Zhu, Thomas Andrus, John McNevin, Jean Lee, James I Mullins, Lawrence Corey, M Juliana McElrath, Tuofu Zhu

  Journal of virology. 09/2009;

  Understanding the mechanisms underlying potential altered susceptibility to HIV-1 infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle E... [more] Understanding the mechanisms underlying potential altered susceptibility to HIV-1 infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1 infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific CTL responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTL, which have been associated with disease control, were detected after but not before seroconversion in LSC63. Furthermore, for the majority of the protein coding regions of HIV-1 variants in LSC63 (except gp41, nef and 3' half of pol), the genetic distances between the infecting and exposed (P63) viruses were comparable to the distances between random subtype B HIV-1 sequences and the exposed (P63) viruses. These results suggest that broad pre-infection immune responses were not able to prevent acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.
• 5.15

  Impact points

  HIV-1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells.

  Brian P. Doehle, Florian Hladik, John P McNevin, M Juliana McElrath, Michael Gale

  Journal of virology. 09/2009;

  Interferon regulatory factor (IRF)-3 is essential for innate intracellular immune defenses that limit virus replication but these defenses fail to suppress HIV infection, which can ultimately associate with opportunistic co-infections and the progression to AIDS. Here, we examined antiviral defenses... [more] Interferon regulatory factor (IRF)-3 is essential for innate intracellular immune defenses that limit virus replication but these defenses fail to suppress HIV infection, which can ultimately associate with opportunistic co-infections and the progression to AIDS. Here, we examined antiviral defenses in CD4+ cells during virus infection and co-infection, revealing that HIV-1 directs a global disruption of innate immune signaling and supports a co-infection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells that supported robust viral replication, whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle, and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+ T cells from acutely HIV-1 infected patients but not from long-term non-progressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus co-infections.
• 8.98

  Impact points

  Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response.

  Ryan M Troyer, John McNevin, Yi Liu, Shao Chong Zhang, Randall W Krizan, Awet Abraha, Denis M Tebit, Hong Zhao, Santiago Avila, Michael A Lobritz, M Juliana McElrath, Sylvie Le Gall, James I Mullins, Eric J Arts

  PLoS pathogens. 05/2009; 5(4):e1000365.

  Human lymphocyte antigen (HLA)-restricted CD8(+) cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escap... [more] Human lymphocyte antigen (HLA)-restricted CD8(+) cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs.

• Escape Mutation in an Immunodominant HIV-1 Tat Epitope Increases Tat Activity

  John McNevin, Jianhong Cao, Matthew McSweyn, M. Juliana McElrath

  AIDS Vaccine 2008, Capetown, SA; 10/2008

  Background One of the major obstacles to maintaining immune control over HIV infection is viral escape mutation. The current dogma suggests that escape mutations will result in a reduction of viral fitness through loss of function. Here, we characterize an immunodominant Tat epitope observed in HIV-... [more] Background One of the major obstacles to maintaining immune control over HIV infection is viral escape mutation. The current dogma suggests that escape mutations will result in a reduction of viral fitness through loss of function. Here, we characterize an immunodominant Tat epitope observed in HIV-1 early infection, subsequent tat sequence evolution, and assess effects on tat transactivation and virion production. Methods PBMCs obtained from Class I HLA Cw*12+ individuals presenting early HIV-1 infection (n=6, range: 31 to 98 days post-acute symptoms) were used in ELISPOT assays to detect CTL responses to a previously described Cw*12-restricted Tat-specific epitope CCFHCQVC (CC8). RNA isolated from PBMCs was used to synthesize cDNA. cDNA was used as template in PCR to amplify and enumerate tat sequences present during early infection. Tat sequences were cloned into an expression vector for comparison of pre-escape, escape, and site-directed mutant tat genes in transactivation and virion production assays Results All 6 Cw*12+ individuals tested had ELISPOT responses to Tat CCFHCQVC. Eighty three per cent (5/6) of individuals had dominant CCFHCQVC sequences at the earliest timepoint; 40% (2/5) of these individuals showed a dominant CCLHCQVC escape mutation subsequently. Minor transient epitopic variants were observed in 67% (4/6) of individuals. One individual, having the latest post-acute infection sampling timepoint, showed CCLHCQVC at all timepoints tested. In transactivation and virion production assays, escape (CCLHCQVC) tat showed higher activity and virion production than pre-escape (CCFHCQVC) tat. This increase in gene function was also assessed and validated by site-directed mutagenesis of tat constructs. Conclusions We have characterized an immunodominant Tat-specific response in early infection, leading to CTL-selected viral escape, and demonstrate that viral immune escape does not necessarily result in reduction of function of the CTL-targeted viral protein.

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• 10.56

  Impact points

  Generation of HIV-1 specific CD8+ cell responses following allogeneic hematopoietic cell transplant.

  Ann E Woolfrey, Uma Malhotra, Robert D. Harrington, John McNevin, Thomas J Manley, Stanley R Riddell, Robert W. Coombs, Frederick R Appelbaum, Larry Corey, Rainer Storb

  Blood. 09/2008;

  This study tested whether donor-derived HIV-specific immune responses could be detected when viral replication was completely suppressed by the continuous administration of highly active anti-retroviral therapy (HAART). A regimen of fludarabine and 200 cGy total body irradiation was followed by infu... [more] This study tested whether donor-derived HIV-specific immune responses could be detected when viral replication was completely suppressed by the continuous administration of highly active anti-retroviral therapy (HAART). A regimen of fludarabine and 200 cGy total body irradiation was followed by infusion of allogeneic donor peripheral blood cells and post-transplant cyclosporine and mycophenolate mofetil. Viral load, lymphocyte counts, and HIV-1-specific CD8+ cell immune responses were compared before and after HCT. Uninterrupted administration of HAART was feasible during nonmyeloablative conditioning and following HCT. The HIV RNA remained undetectable and no HIV-associated infections were observed. CD8+ T-cell responses targeting multiple epitopes were detected prior to HCT. Following HCT a different pattern of donor-derived HIV-specific CTL responses emerged by day +80, presumably primed in vivo. We conclude that allogeneic HCT offers the unique ability to characterize de novo HIV-1-specific immune responses. This clinical trial was registered at ClinicalTrials.gov Identifier: NCT00112593.
• 5.15

  Impact points

  Novel cytotoxic T-lymphocyte escape mutation by a three-amino-acid insertion in the human immunodeficiency virus type 1 p6Pol and p6Gag late domain associated with drug resistance.

  Jianhong Cao, John McNevin, Matthew McSweyn, Yi Liu, James I Mullins, M Juliana McElrath

  Journal of virology. 02/2008; 82(1):495-502.

  Cytolytic T lymphocytes (CTL) play a major role in controlling human immunodeficiency virus type 1 (HIV-1) infection. To evade immune pressure, HIV-1 is selected at targeted CTL epitopes, which may consequentially alter viral replication fitness. In our longitudinal investigations of the interplay b... [more] Cytolytic T lymphocytes (CTL) play a major role in controlling human immunodeficiency virus type 1 (HIV-1) infection. To evade immune pressure, HIV-1 is selected at targeted CTL epitopes, which may consequentially alter viral replication fitness. In our longitudinal investigations of the interplay between T-cell immunity and viral evolution following acute HIV-1 infection, we observed in a treatment-naïve patient the emergence of highly avid, gamma interferon-secreting, CD8(+) CTL recognizing an HLA-Cw*0102-restricted epitope, NSPTRREL (NL8). This epitope lies in the p6(Pol) protein, located in the transframe region of the Gag-Pol polyprotein. Over the course of infection, an unusual viral escape mutation arose within the p6(Pol) epitope through insertion of a 3-amino-acid repeat, NSPT(SPT)RREL, with a concomitant insertion in the p6(Gag) late domain, PTAPP(APP). Interestingly, this p6(Pol) insertion mutation is often selected in viruses with the emergence of antiretroviral drug resistance, while the p6(Gag) late-domain PTAPP motif binds Tsg101 to permit viral budding. These results are the first to demonstrate viral evasion of immune pressure by amino acid insertions. Moreover, this escape mutation represents a novel mechanism whereby HIV-1 can alter its sequence within both the Gag and Pol proteins with potential functional consequences for viral replication and budding.
• 5.15

  Impact points

  Evolution of human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitopes: fitness-balanced escape.

  Yi Liu, John McNevin, Hong Zhao, Denis M Tebit, Ryan M Troyer, Matthew McSweyn, Ananta K Ghosh, Daniel Shriner, Eric J Arts, M Juliana McElrath, James I Mullins

  Journal of virology. 12/2007; 81(22):12179-88.

  CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present a... [more] CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.
• 5.65

  Impact points

  Preservation of T cell proliferation restricted by protective HLA alleles is critical for immune control of HIV-1 infection.

  Helen Horton, Ian Frank, Ruth Baydo, Emilie Jalbert, Justin Penn, Sean Wilson, John P McNevin, Matthew D McSweyn, Deborah Lee, Yunda Huang, Stephen C De Rosa, M Juliana McElrath

  Journal of immunology (Baltimore, Md. : 1950). 12/2006; 177(10):7406-15.

  HIV-1-infected persons with HLA-B27 and -B57 alleles commonly remain healthy for decades without antiretroviral therapy. Properties of CD8+ T cells restricted by these alleles considered to confer disease protection in these individuals are elusive but important to understand and potentially elicit ... [more] HIV-1-infected persons with HLA-B27 and -B57 alleles commonly remain healthy for decades without antiretroviral therapy. Properties of CD8+ T cells restricted by these alleles considered to confer disease protection in these individuals are elusive but important to understand and potentially elicit by vaccination. To address this, we compared CD8+ T cell function induced by HIV-1 immunogens and natural infection using polychromatic flow cytometry. HIV-1-specific CD8+ T cells from all four uninfected immunized and 21 infected subjects secreted IFN-gamma and TNF-alpha. However, CD8+ T cells induced by vaccination and primary infection, but not chronic infection, proliferated to their cognate epitopes. Notably, B27- and B57-restricted CD8+ T cells from nonprogressors exhibited greater expansion than those restricted by other alleles. Hence, CD8+ T cells restricted by certain protective alleles can resist replicative defects, which permits expansion and antiviral effector activities. Our findings suggest that the capacity to maintain CD8+ T cell proliferation, regardless of MHC-restriction, may serve as an important correlate of disease protection in the event of infection following vaccination.
• 5.15

  Impact points

  Selection on the human immunodeficiency virus type 1 proteome following primary infection.

  Yi Liu, John McNevin, Jianhong Cao, Hong Zhao, Indira Genowati, Kim Wong, Sherry McLaughlin, Matthew D McSweyn, Kurt Diem, Claire E Stevens, Janine Maenza, Hongxia He, David C Nickle, Daniel Shriner, Sarah E Holte, Ann C Collier, Lawrence Corey, M Juliana McElrath, James I Mullins

  Journal of virology. 11/2006; 80(19):9519-29.

  Typically during human immunodeficiency virus type 1 (HIV-1) infection, a nearly homogeneous viral population first emerges and then diversifies over time due to selective forces that are poorly understood. To identify these forces, we conducted an intensive longitudinal study of viral genetic chang... [more] Typically during human immunodeficiency virus type 1 (HIV-1) infection, a nearly homogeneous viral population first emerges and then diversifies over time due to selective forces that are poorly understood. To identify these forces, we conducted an intensive longitudinal study of viral genetic changes and T-cell immunity in one subject at < or =17 time points during his first 3 years of infection, and in his infecting partner near the time of transmission. Autologous peptides covering amino acid sites inferred to be under positive selection were powerful for identifying HIV-1-specific cytotoxic-T-lymphocyte (CTL) epitopes. Positive selection and mutations resulting in escape from CTLs occurred across the viral proteome. We detected 25 CTL epitopes, including 14 previously unreported. Seven new epitopes mapped to the viral Env protein, emphasizing Env as a major target of CTLs. One-third of the selected sites were associated with epitopic mutational escapes from CTLs. Most of these resulted from replacement with amino acids found at low database frequency. Another one-third represented acquisition of amino acids found at high database frequency, suggesting potential reversions of CTL epitopic sites recognized by the immune system of the transmitting partner and mutation toward improved viral fitness in the absence of immune targeting within the recipient. A majority of the remaining selected sites occurred in the envelope protein and may have been subjected to humoral immune selection. Hence, a majority of the amino acids undergoing selection in this subject appeared to result from fitness-balanced CTL selection, confirming CTLs as a dominant selective force in HIV-1 infection.
• 5.15

  Impact points

  Induction of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses in HIV vaccine trial participants who subsequently acquire HIV-1 infection.

  Helen Horton, Colin Havenar-Daughton, Deborah Lee, Erin Moore, Jianhong Cao, John McNevin, Thomas Andrus, Haiying Zhu, Abbe Rubin, Tuofu Zhu, Connie Celum, M Juliana McElrath

  Journal of virology. 11/2006; 80(19):9779-88.

  Candidate human immunodeficiency virus type 1 (HIV-1) vaccines designed to elicit T-cell immunity in HIV-1-uninfected persons are under investigation in phase I to III clinical trials. Little is known about how these vaccines impact the immunologic response postinfection in persons who break through... [more] Candidate human immunodeficiency virus type 1 (HIV-1) vaccines designed to elicit T-cell immunity in HIV-1-uninfected persons are under investigation in phase I to III clinical trials. Little is known about how these vaccines impact the immunologic response postinfection in persons who break through despite vaccination. Here, we describe the first comprehensive characterization of HIV-specific T-cell immunity in vaccine study participants following breakthrough HIV-1 infection in comparison to 16 nonvaccinated subjects with primary HIV-1 infection. Whereas none of the 16 breakthrough infections possessed vaccine-induced HIV-1-specific T-cell responses preinfection, 85% of vaccinees and 86% of nonvaccinees with primary HIV-1 infection developed HIV-specific T-cell responses postinfection. Breakthrough subjects' T cells recognized 43 unique HIV-1 T-cell epitopes, of which 8 are newly described, and 25% were present in the vaccine. The frequencies of gamma interferon (IFN-gamma)-secreting cells recognizing epitopes within gene products that were and were not encoded by the vaccine were not different (P = 0.64), which suggests that responses were not anamnestic. Epitopes within Nef and Gag proteins were the most commonly recognized in both vaccinated and nonvaccinated infected subjects. One individual controlled viral replication without antiretroviral therapy and, notably, mounted a novel HIV-specific HLA-C14-restricted Gag LYNTVATL-specific T-cell response. Longitudinally, HIV-specific T cells in this individual were able to secrete IFN-gamma and tumor necrosis factor alpha, as well as proliferate and degranulate in response to their cognate antigenic peptides up to 5 years postinfection. In conclusion, a vaccinee's ability to mount an HIV-specific T-cell response postinfection is not compromised by previous immunization, since the CD8+ T-cell responses postinfection are similar to those seen in vaccine-naïve individuals. Finding an individual who is controlling infection highlights the importance of comprehensive studies of breakthrough infections in vaccine trials to determine whether host genetics/immune responses and/or viral characteristics are responsible for controlling viral replication.
• 5.65

  Impact points

  Evolution of CD8+ T cell immunity and viral escape following acute HIV-1 infection.

  Jianhong Cao, John McNevin, Uma Malhotra, M Juliana McElrath

  Journal of immunology (Baltimore, Md. : 1950). 11/2003; 171(7):3837-46.

  Induction of HIV-1-specific CD8(+) T cells during acute infection is associated with a decline in viremia. The role CD8(+) effectors play in subsequently establishing viral set point remains unclear. To address this, we focused on two acutely infected patients with the same initial Tat-specific CD8(... [more] Induction of HIV-1-specific CD8(+) T cells during acute infection is associated with a decline in viremia. The role CD8(+) effectors play in subsequently establishing viral set point remains unclear. To address this, we focused on two acutely infected patients with the same initial Tat-specific CD8(+) response, analyzing their CD8(+) T cell responses longitudinally in conjunction with viral load and sequence evolution. In one patient initiating treatment during acute infection, the frequencies of Tat-specific CD8(+) T cells gradually diminished but persisted, and the Tat epitope sequence was unaltered. By contrast, in the second patient who declined treatment, the Tat-specific CD8(+) T cells disappeared below detection, in conjunction with Gag-specific CD4(+) T cell loss, as plasma viremia reached a set point. This coincided with the emergence of an escape variant within the Tat epitope and an additional Vpr epitope. New CD8(+) T cell responses emerged but with no further associated decline in viremia. These findings indicate that, in the absence of treatment, the initial CD8(+) T cell responses have the greatest impact on reducing viremia, and that later, continuously evolving responses are less efficient in further reducing viral load. The results also suggest that T cell help may contribute to the antiviral efficiency of the acute CD8(+) T cell response.
• 5.15

  Impact points

  Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-secreting CD8+ T cells in primary HIV-1 infection.

  Jianhong Cao, John McNevin, Sarah Holte, Lisa Fink, Lawrence Corey, M Juliana McElrath

  Journal of virology. 06/2003; 77(12):6867-78.

  Human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we d... [more] Human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we determined the fine specificities and frequencies of gamma interferon (IFN-gamma)-secreting CD8(+) T cells recognizing all HIV-1 proteins in patients with primary infection. In these subjects, the earliest detected responses were directed predominantly against Nef, Tat, Vpr, and Env. Tat- and Vpr-specific CD8(+) T cells accounted for the greatest frequencies of mean IFN-gamma spot-forming cells (SFC). Nef-specific responses (10 of 21) were more commonly detected. A mean of 2.3 epitopes were recognized with various avidities per subject, and the number increased with the duration of infection (R = 0.47, P = 0.031). The mean frequency of CD8(+) T cells (985 SFC/10(6) peripheral blood mononuclear cells) correlated with the number of epitopes recognized (R = 0.84, P < 0.0001) and the number of HLA-restricting alleles (R = 0.79, P < 0.0001). Neither the total SFC frequencies nor the number of epitopes recognized correlated with the concurrent plasma viral load. Seventeen novel epitopes were identified, four of which were restricted to HLA alleles (A23 and B72) that are common among African descendents. Thus, primary HIV-1 infection induces strong CD8(+)-T-cell immunity whose specificities broaden over time, but their frequencies and breadth do not correlate with HIV-1 containment when examined concurrently. Many novel epitopes, particularly directed to Nef, Tat, and Env, and frequently with unique HLA restrictions, merit further consideration in vaccine design.
• 4.16

  Impact points

  Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology.

  W K Fong, Z Modrusan, J P McNevin, J Marostenmaki, B Zin, F Bekkaoui

  Journal of clinical microbiology. 08/2000; 38(7):2525-9.

  A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3&#... [more] A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3' terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the mecA gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the mecA gene, the probe is cut and no test line is formed on the strip. A screen of 324 S. aureus clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the mecA gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of mecA-negative non-S. aureus isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the mecA gene in S. aureus isolates.
• 2.45

  Impact points

  Rapid detection of the mecA gene in methicillin resistant staphylococci using a colorimetric cycling probe technology.

  F Bekkaoui, J P McNevin, C H Leung, G J Peterson, A Patel, R S Bhatt, R N Bryan

  Diagnostic microbiology and infectious disease. 07/1999; 34(2):83-90.

  A Cycling Probe Technology (CPT) assay was developed for the detection of the mecA gene from methicillin resistant staphylococcal cultures. The assay is based on a colorimetric enzyme-immuno-assay (EIA) and uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5'-terminus and biotin at... [more] A Cycling Probe Technology (CPT) assay was developed for the detection of the mecA gene from methicillin resistant staphylococcal cultures. The assay is based on a colorimetric enzyme-immuno-assay (EIA) and uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5'-terminus and biotin at the 3'-terminus. The reaction occurs at a constant temperature that allows the target DNA to anneal to the probe. RNase H cuts the RNA portion, allowing the cut fragments to dissociate from the target, making it available for further cycling. CPT-EIA uses streptavidin-coated microplate wells to capture uncut probe followed by detection with horseradish-peroxidase conjugated anti-fluorescein antibody. The assay was compared to PCR and shown to accurately detect the presence or absence of the mecA gene in 159 staphylococcal clinical isolates. The CPT-EIA assay takes two hours starting from cultured cells compared with the 24-48 h required for detection of methicillin resistance by conventional susceptibility tests.