Saverio Capuano

Subdominant CD8+ T-Cell Responses Are Involved in Durable Control of AIDS Virus Replication

ABSTRACT “Elite controllers” are individuals that durably control human immunodeficiency virus or simian immunodeficiency virus replication without therapeutic intervention. The study of these rare individuals may facilitate the definition of a successful immune response to immunodeficiency viruses. Here we describe six Indian-origin rhesus macaques that have controlled replication of the pathogenic virus SIVmac239 for 1 to 5 years. To determine which lymphocyte populations were responsible for this control, we transiently depleted the animals’ CD8+ cells in vivo. This treatment resulted in 100- to 10,000-fold increases in viremia. When the CD8+ cells returned, control was reestablished and the levels of small subsets of previously subdominant CD8+ T cells expanded up to 2,500-fold above predepletion levels. This wave of CD8+ T cells was accompanied by robust Gag-specific CD4 responses. In contrast, CD8+ NK cell frequencies changed no more than threefold. Together, our data suggest that CD8+ T cells targeting a small number of epitopes, along with broad CD4+ T-cell responses, can successfully control the replication of the AIDS virus. It is likely that subdominant CD8+ T-cell populations play a key role in maintaining this control.

A rhesus macaque model of Asian-lineage Zika virus infection

Infection with Asian-lineage Zika virus (ZIKV) has been associated with Guillain–Barré syndrome and fetal abnormalities, but the underlying mechanisms remain poorly understood. Animal models of infection are thus urgently needed. Here we show that rhesus macaques are susceptible to infection by an Asian-lineage ZIKV closely related to strains currently circulating in the Americas. Following subcutaneous inoculation, ZIKV RNA is detected in plasma 1 day post infection (d.p.i.) in all animals (N=8, including 2 pregnant animals), and is also present in saliva, urine and cerebrospinal fluid. Non-pregnant and pregnant animals remain viremic for 21 days and for up to at least 57 days, respectively. Neutralizing antibodies are detected by 21 d.p.i. Rechallenge 10 weeks after the initial challenge results in no detectable virus replication, indicating protective immunity against homologous strains. Therefore, Asian-lineage ZIKV infection of rhesus macaques provides a relevant animal model for studying pathogenesis and evaluating potential interventions against human infection, including during pregnancy.

Vaccine-induced CD8+ T cells control AIDS virus replication

Developing a vaccine for human immunodeficiency virus (HIV) may be aided by a complete understanding of those rare cases in which some HIV-infected individuals control replication of the virus. Most of these elite controllers express the histocompatibility alleles HLA-B*57 or HLA-B*27 (ref. 3). These alleles remain by far the most robust associations with low concentrations of plasma virus, yet the mechanism of control in these individuals is not entirely clear. Here we vaccinate Indian rhesus macaques that express Mamu-B*08, an animal model for HLA-B*27-mediated elite control, with three Mamu-B*08-restricted CD8+ T-cell epitopes, and demonstrate that these vaccinated animals control replication of the highly pathogenic clonal simian immunodeficiency virus (SIV) mac239 virus. High frequencies of CD8+ T cells against these Vif and Nef epitopes in the blood, lymph nodes and colon were associated with viral control. Moreover, the frequency of the CD8+ T-cell response against the Nef RL10 epitope (Nef amino acids 137–146) correlated significantly with reduced acute phase viraemia. Finally, two of the eight vaccinees lost control of viral replication in the chronic phase, concomitant with escape in all three targeted epitopes, further implicating these three CD8+ T-cell responses in the control of viral replication. Our findings indicate that narrowly targeted vaccine-induced virus-specific CD8+ T-cell responses can control replication of the AIDS virus.

Cross-reactive T cells are involved in rapid clearance of 2009 pandemic H1N1 influenza virus in nonhuman primates.

In mouse models of influenza, T cells can confer broad protection against multiple viral subtypes when antibodies raised against a single subtype fail to do so. However, the role of T cells in protecting humans against influenza remains unclear. Here we employ a translational nonhuman primate model to show that cross-reactive T cell responses play an important role in early clearance of infection with 2009 pandemic H1N1 influenza virus (H1N1pdm). To “prime” cellular immunity, we first infected 5 rhesus macaques with a seasonal human H1N1 isolate. These animals made detectable cellular and antibody responses against the seasonal H1N1 isolate but had no neutralizing antibodies against H1N1pdm. Four months later, we challenged the 5 “primed” animals and 7 naive controls with H1N1pdm. In naive animals, CD8+ T cells with an activated phenotype (Ki-67+ CD38+) appeared in blood and lung 5–7 days post inoculation (p.i.) with H1N1pdm and reached peak magnitude 7–10 days p.i. In contrast, activated T cells were recruited to the lung as early as 2 days p.i. in “primed” animals, and reached peak frequencies in blood and lung 4–7 days p.i. Interferon (IFN)-γ Elispot and intracellular cytokine staining assays showed that the virus-specific response peaked earlier and reached a higher magnitude in “primed” animals than in naive animals. This response involved both CD4+ and CD8+ T cells. Strikingly, “primed” animals cleared H1N1pdm infection significantly earlier from the upper and lower respiratory tract than the naive animals did, and before the appearance of H1N1pdm-specific neutralizing antibodies. Together, our results suggest that cross-reactive T cell responses can mediate early clearance of an antigenically novel influenza virus in primates. Vaccines capable of inducing such cross-reactive T cells may help protect humans against severe disease caused by newly emerging pandemic influenza viruses.

Highly efficient maternal-fetal Zika virus transmission in pregnant rhesus macaques

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10–12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.

Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques

Background Zika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV. Methodology/Principal Findings Using our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form. Conclusions/Significance An effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains.

Recombinant Yellow Fever Vaccine Virus 17D Expressing Simian Immunodeficiency Virus SIVmac239 Gag Induces SIV-Specific CD8+ T-Cell Responses in Rhesus Macaques

ABSTRACT Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8+ T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8+ T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8+ T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4+ T cells.

Avian-Type Receptor-Binding Ability Can Increase Influenza Virus Pathogenicity in Macaques

ABSTRACT The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.

A Novel Nonhuman Primate Model for Influenza Transmission

Studies of influenza transmission are necessary to predict the pandemic potential of emerging influenza viruses. Currently, both ferrets and guinea pigs are used in such studies, but these species are distantly related to humans. Nonhuman primates (NHP) share a close phylogenetic relationship with humans and may provide an enhanced means to model the virological and immunological events in influenza virus transmission. Here, for the first time, it was demonstrated that a human influenza virus isolate can productively infect and be transmitted between common marmosets (Callithrix jacchus), a New World monkey species. We inoculated four marmosets with the 2009 pandemic virus A/California/07/2009 (H1N1pdm) and housed each together with a naïve cage mate. We collected bronchoalveolar lavage and nasal wash samples from all animals at regular intervals for three weeks post-inoculation to track virus replication and sequence evolution. The unadapted 2009 H1N1pdm virus replicated to high titers in all four index animals by 1 day post-infection. Infected animals seroconverted and presented human-like symptoms including sneezing, nasal discharge, labored breathing, and lung damage. Transmission occurred in one cohabitating pair. Deep sequencing detected relatively few genetic changes in H1N1pdm viruses replicating in any infected animal. Together our data suggest that human H1N1pdm viruses require little adaptation to replicate and cause disease in marmosets, and that these viruses can be transmitted between animals. Marmosets may therefore be a viable model for studying influenza virus transmission.

Oropharyngeal mucosal transmission of Zika virus in rhesus macaques

Zika virus is present in urine, saliva, tears, and breast milk, but the transmission risk associated with these body fluids is currently unknown. Here we evaluate the risk of Zika virus transmission through mucosal contact in rhesus macaques. Application of high-dose Zika virus directly to the tonsils of three rhesus macaques results in detectable plasma viremia in all animals by 2 days post-exposure; virus replication kinetics are similar to those observed in animals infected subcutaneously. Three additional macaques inoculated subcutaneously with Zika virus served as saliva donors to assess the transmission risk from contact with oral secretions from an infected individual. Seven naive animals repeatedly exposed to donor saliva via the conjunctivae, tonsils, or nostrils did not become infected. Our results suggest that there is a risk of Zika virus transmission via the mucosal route, but that the risk posed by oral secretions from individuals with a typical course of Zika virus infection is low.Zika virus (ZIKV) is present in body fluids, including saliva, but transmission risk through mucosal contact is not well known. Here, the authors show that oropharyngeal mucosal infection of macaques with a high ZIKV dose results in viremia, but that transmission risk from saliva of infected animals is low.