Thakshila Amarasena

MAIT cells are depleted early but retain functional cytokine expression in HIV infection

Mucosal‐associated invariant T (MAIT) cells home to mucosal sites and exert antimicrobial activity against bacteria and other microorganisms. HIV infection leads to early depletion of gut T cells and translocation of bacterial products. There are reports that MAIT cells, defined by coexpression of Vα7.2 and CD161, are depleted during HIV infection and residual MAIT cells are functionally impaired. However, one study suggested that MAIT cells might remain after HIV infection but evade detection through CD161 downregulation. Thus, the impact of HIV infection on MAIT cells is unclear. We studied longitudinal blood samples from 31 HIV‐infected subjects for MAIT cell numbers, phenotype and function using both standard Vα7.2/CD161 surface markers and an MR1 tetramer. We found that MAIT cells were depleted early during HIV infection, and although there was a concomitant rise in Vα7.2+CD161– cells, these were MR1 tetramer negative, indicating that these are unlikely to be altered MAIT cells. Antigen‐mediated activation of residual MAIT cells showed that they remained functional out to 2 years following HIV infection. Although MAIT cells are depleted in HIV infection, residual and functionally active MAIT cells persist and may still be able to assist in controlling bacterial translocation during HIV infection.

Age-associated cross-reactive antibody-dependent cellular cytotoxicity toward 2009 pandemic influenza A virus subtype H1N1.

BACKGROUND During the 2009 pandemic of influenza A virus subtype H1N1 (A[H1N1]pdm09) infection, older individuals were partially protected from severe disease. It is not known whether preexisting antibodies with effector functions such as antibody-dependent cellular cytotoxicity (ADCC) contributed to the immunity observed. METHODS We tested serum specimens obtained from 182 individuals aged 1-72 years that were collected either immediately before or after the A(H1N1)pdm09 pandemic for ADCC antibodies to the A(H1N1)pdm09 hemagglutinin (HA) protein. RESULTS A(H1N1)pdm09 HA-specific ADCC antibodies were detected in almost all individuals aged >45 years (28/31 subjects) before the 2009 A(H1N1) pandemic. Conversely, only approximately half of the individuals aged 1-14 years (11/31) and 15-45 years (17/31) had cross-reactive ADCC antibodies before the 2009 A(H1N1) pandemic. The A(H1N1)pdm09-specific ADCC antibodies were able to efficiently mediate the killing of influenza virus-infected respiratory epithelial cells. Further, subjects >45 years of age had higher ADCC titers to a range of seasonal H1N1 HA proteins, including from the 1918 virus, compared with younger individuals. CONCLUSIONS ADCC antibodies may have contributed to the protection exhibited in older individuals during the 2009 A(H1N1) pandemic. This work has significant implications for improved vaccination strategies for future influenza pandemics.

Standard Trivalent Influenza Virus Protein Vaccination Does Not Prime Antibody-Dependent Cellular Cytotoxicity in Macaques

ABSTRACT Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended, since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce antibody-dependent cellular cytotoxicity (ADCC) responses that can cross-recognize divergent influenza virus strains is unknown. We immunized 6 influenza-naive pigtail macaques twice with the 2011–2012 season TIV and then challenged the macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza virus-specific CD8 T cell (CTL) responses using a sensitive major histocompatibility complex (MHC) tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA), did not induce detectable influenza virus-specific ADCC or CTL responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins, but not influenza virus HA proteins from different subtypes (H2 to H7). There was no anamnestic influenza virus-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or to divergent H3 proteins but did boost CTL responses. ADCC or CTL responses were not induced by TIV vaccination in influenza-naive macaques. There was a marked difference in the ability of infection compared to that of vaccination to induce cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.

Evolution of atypical enteropathogenic E. coli by repeated acquisition of LEE pathogenicity island variants

Atypical enteropathogenic Escherichia coli (aEPEC) is an umbrella term given to E. coli that possess a type III secretion system encoded in the locus of enterocyte effacement (LEE), but lack the virulence factors (stx, bfpA) that characterize enterohaemorrhagic E. coli and typical EPEC, respectively. The burden of disease caused by aEPEC has recently increased in industrialized and developing nations, yet the population structure and virulence profile of this emerging pathogen are poorly understood. Here, we generated whole-genome sequences of 185 aEPEC isolates collected during the Global Enteric Multicenter Study from seven study sites in Asia and Africa, and compared them with publicly available E. coli genomes. Phylogenomic analysis revealed ten distinct widely distributed aEPEC clones. Analysis of genetic variation in the LEE pathogenicity island identified 30 distinct LEE subtypes divided into three major lineages. Each LEE lineage demonstrated a preferred chromosomal insertion site and different complements of non-LEE encoded effector genes, indicating distinct patterns of evolution of these lineages. This study provides the first detailed genomic framework for aEPEC in the context of the EPEC pathotype and will facilitate further studies into the epidemiology and pathogenicity of EPEC by enabling the detection and tracking of specific clones and LEE variants.

Thrombocytopenia Is Strongly Associated With Simian AIDS in Pigtail Macaques

Simian AIDS has a variable time course and presentation making it difficult to define disease effects of progressive simian immunodeficiency virus (SIV) infection. We commonly observed thrombocytopenia (TCP) associated with progressive SIV infection of pigtail macaques (Macaca nemestrina). We therefore analyzed the relationship between platelet counts, viral load (VL), and CD4 T-cell levels in 44 unselected macaques with chronic SIV infection. Persistent TCP was observed in 70% of pigtail macaques infected with SIVmac251 for up to 77 weeks in the absence of clinically significant bleeding. The presence of TCP correlated with higher SIV plasma VLs and depressed total and memory CD4 T cells. TCP was more common in macaques requiring euthanasia for incipient AIDS than macaques that survived to the end of the studies, although VL and CD4 T-cell decline were stronger independent predictors of AIDS-free survival. There was however no clear correlation between the development of TCP and immune activation as measured by plasma soluble CD14. We conclude that TCP is a useful end point to analyze SIV studies in pigtail macaques.

Safety, immunogenicity and efficacy of peptide-pulsed cellular immunotherapy in macaques

Background  Simple and effective delivery methods for cellular immunotherapies are needed. We recently published on the effectiveness of using ex vivo pulsing of overlapping SIV Gag 15mer peptides onto fresh peripheral blood cells in 32 SIVmac251‐infected pigtail macaques.

Partial efficacy of a broadly neutralizing antibody against cell-associated SHIV infection

Broadly neutralizing antibody confers partial protection from cell-associated SHIV. Antibodies circumvented by cell-associated virus Antibodies that are able to neutralize a range of HIV-1 strains are being developed to prevent infection in humans. Preclinical testing of these antibodies predominantly uses free virus during the challenge, although natural HIV-1 transmission could involve transfer of infected cells. To determine whether antibodies would still be able to combat this type of transmission, Parsons et al. developed a challenge model in nonhuman primates using SHIV-infected splenocytes. A well-characterized anti–HIV-1 antibody failed to protect all of the animals from the cell-associated viral challenge, although it had protected all animals from cell-free viral challenge. Their findings indicate that sustained high concentrations of circulating antibody may be necessary for prevention of cell-associated HIV-1 acquisition. Broadly neutralizing antibodies (BnAbs) protect macaques from cell-free simian/human immunodeficiency virus (SHIV) challenge, but their efficacy against cell-associated SHIV is unclear. Virus in cell-associated format is highly infectious, present in transmission-competent bodily fluids, and potentially capable of evading antibody-mediated neutralization. The PGT121 BnAb, which recognizes an epitope consisting of the V3 loop and envelope glycans, mediates antibody-dependent cellular cytotoxicity and neutralization of cell-to-cell HIV-1 transmission. To evaluate whether a BnAb can prevent infection after cell-associated viral challenge, we infused pigtail macaques with PGT121 or an isotype control and challenged animals 1 hour later intravenously with SHIVSF162P3-infected splenocytes. All five controls had high viremia 1 week after challenge. Three of six PGT121-infused animals were completely protected, two of six animals had a 1-week delay in onset of high viremia, and one animal had a 7-week delay in onset of viremia. The infused antibody had decayed on average to 2.0 μg/ml by 1 week after infusion and was well below 1 μg/ml (range, <0.1 to 0.8 μg/ml) by 8 weeks. The animals with a 1-week delay before high viremia had relatively lower plasma concentrations of PGT121. Transfer of 22 million peripheral blood mononuclear cells (PBMCs) stored at weeks 1 to 4 from the animal with the 7-week delayed onset of viremia into uninfected macaques did not initiate infection. Our results show that HIV-1–specific neutralizing antibodies have partial efficacy against cell-associated virus exposure in macaques. We conclude that sustaining high concentrations of bioavailable BnAb is important for protecting against cell-associated virus.

Simian immunodeficiency virus infection and immune responses in the pig-tailed macaque testis

The testis is a site of immune privilege in rodents, and there is evidence that T cell responses are also suppressed in the primate testis. Local immunosuppression is a potential mechanism for HIV persistence in tissue reservoirs that few studies have examined. The response of the pig‐tailed macaque testis to SIVmac239 infection was characterized to test this possibility. Testes were surgically removed during early‐chronic (10 wk) and late‐chronic (24–30 wk) SIV infection in 4 animals and compared with those from 7 uninfected animals. SIV infection caused only minor disruption to the seminiferous epithelium without marked evidence of inflammation or consistent changes in total intratesticular leukocyte numbers. Infection also led to an increase in the relative proportion of testicular effector memory CD8+ T cell numbers and a corresponding reduction in central memory CD4+ T cells. A decrease in the relative proportion of resident‐type CD163+ macrophages and DCs was also observed. SIV‐specific CD8+ T cells were detectable in the testis, 10–11 wk after infection by staining with SIV Gag‐specific or Tat‐specific MHC‐I tetramers. However, testicular CD8+ T cells from the infected animals had suppressed cytokine responses to mitogen activation. These results support the possibility that local immunosuppression in the testis may be restricting the ability of T cells to respond to SIV or HIV infection. Local immunosuppression in the testis may be an underexplored mechanism allowing HIV persistence.

Recombinant influenza virus expressing HIV-1 p24 capsid protein induces mucosal HIV-specific CD8 T-cell responses.

Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection.

High fidelity simian immunodeficiency virus reverse transcriptase mutants have impaired replication in vitro and in vivo.

The low fidelity of HIV replication facilitates immune and drug escape. Some reverse transcriptase (RT) inhibitor drug-resistance mutations increase RT fidelity in biochemical assays but their effect during viral replication is unclear. We investigated the effect of RT mutations K65R, Q151N and V148I on SIV replication and fidelity in vitro, along with SIV replication in pigtailed macaques. SIVmac239-K65R and SIVmac239-V148I viruses had reduced replication capacity compared to wild-type SIVmac239. Direct virus competition assays demonstrated a rank order of wild-type>K65R>V148I mutants in terms of viral fitness. In single round in vitro-replication assays, SIVmac239-K65R demonstrated significantly higher fidelity than wild-type, and rapidly reverted to wild-type following infection of macaques. In contrast, SIVmac239-Q151N was replication incompetent in vitro and in pigtailed macaques. Thus, we showed that RT mutants, and specifically the common K65R drug-resistance mutation, had impaired replication capacity and higher fidelity. These results have implications for the pathogenesis of drug-resistant HIV.