Sallie R. Permar

Pathogenic Simian Immunodeficiency Virus Infection Is Associated with Expansion of the Enteric Virome

Pathogenic simian immunodeficiency virus (SIV) infection is associated with enteropathy, which likely contributes to AIDS progression. To identify candidate etiologies for AIDS enteropathy, we used next-generation sequencing to define the enteric virome during SIV infection in nonhuman primates. Pathogenic, but not nonpathogenic, SIV infection was associated with significant expansion of the enteric virome. We identified at least 32 previously undescribed enteric viruses during pathogenic SIV infection and confirmed their presence by using viral culture and PCR testing. We detected unsuspected mucosal adenovirus infection associated with enteritis as well as parvovirus viremia in animals with advanced AIDS, indicating the pathogenic potential of SIV-associated expansion of the enteric virome. No association between pathogenic SIV infection and the family-level taxonomy of enteric bacteria was detected. Thus, enteric viral infections may contribute to AIDS enteropathy and disease progression. These findings underline the importance of metagenomic analysis of the virome for understanding AIDS pathogenesis.

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.

Conditions that may affect the results of susceptibility testing of Mycobacterium tuberculosis to pyrazinamide.

Pyrazinamide (PZA) is an important front-line anti-tuberculosis drug that is active only at acid pH. However, acid pH causes significant difficulty for PZA susceptibility testing. A common problem in PZA testing is false resistance caused by large bacterial inocula. This study investigated the relationship of false resistance to numbers of bacilli, pH and other factors that potentially affect susceptibility to PZA. Large inocula (10(7-8) bacilli/ml) of M. tuberculosis H37Ra caused significant increase in medium pH from 5.5 towards neutrality, and thus produced false resistance results. The increase in medium pH was determined to be a function of live bacilli; heat-killed bacilli had little or no effect. Susceptibility to PZA and its active derivative pyrazinoic acid (POA) was comparable on 7H11 agar medium, but POA was less active than PZA in liquid medium containing bovine serum albumin (BSA), suggesting that susceptibility to PZA or POA was reduced in the presence of BSA, because of its neutralising effect on medium pH and significant POA binding. A 3-month-old H37Ra culture was shown to be more susceptible to PZA exposure than a 4-day log-phase culture, suggesting that PZA is more active for non-growing bacilli. Finally, reserpine, an inhibitor of POA efflux pump, increased susceptibility to PZA even near neutral pH 6.8, with an MIC of 400 mg/L compared with 1,000 mg/L without reserpine. These findings should have implications for understanding the mode of action of PZA and for PZA susceptibility testing.

Successful DNA immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles

Measles remains a principal cause of worldwide mortality, in part because young infants cannot be immunized effectively. Development of new vaccines has been hindered by previous experience with a formalin-inactivated vaccine that predisposed to a severe form of disease (atypical measles). Here we have developed and tested potential DNA vaccines for immunogenicity, efficacy and safety in a rhesus macaque model of measles. DNA protected from challenge with wild-type measles virus. Protection correlated with levels of neutralizing antibody and not with cytotoxic T lymphocyte activity. There was no evidence in any group, including those receiving hemagglutinin-encoding DNA alone, of ‘priming’ for atypical measles.

Transmitted/Founder and Chronic Subtype C HIV-1 Use CD4 and CCR5 Receptors with Equal Efficiency and Are Not Inhibited by Blocking the Integrin α4β7

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.

Prolonged Measles Virus Shedding in Human Immunodeficiency Virus—Infected Children, Detected by Reverse Transcriptase—Polymerase Chain Reaction

A reverse transcriptase-polymerase chain reaction assay was used to detect measles virus RNA in peripheral blood mononuclear cells, urine, and nasopharyngeal specimens from Zambian children during hospitalization and approximately 1-2 months after discharge. Of 47 children, 29 (61.7%) had prolonged measles virus shedding, as defined by detection of measles virus RNA in > or =1 specimen obtained 30-61 days after rash onset. Ten (90.9%) of 11 human immunodeficiency virus (HIV)-infected children had prolonged measles virus shedding, compared with 19 (52.8%) of 36 HIV-uninfected children (P=.02). Prolonged measles virus shedding did not correlate with levels of measles virus-specific antibody. HIV-infected children with measles may have a prolonged infectious period that potentially enhances measles virus transmission and hinders measles control.

Limited Contribution of Humoral Immunity to the Clearance of Measles Viremia in Rhesus Monkeys

The development of an improved vaccine for controlling measles virus (MV) infections in the developing world will require an understanding of the immune mechanisms responsible for the clearance of this virus. To evaluate the role of humoral immunity in the containment of MV, rhesus monkeys were treated at the time of MV challenge with either anti-CD20 monoclonal antibody (MAb) infusion, to deplete B lymphocytes, or both anti-CD20 and anti-CD8 MAb, to deplete both B lymphocytes and CD8+ effector T lymphocytes. Although the MV-specific antibody response in CD20+ lymphocyte-depleted monkeys was delayed by >1 week, the kinetics of MV clearance did not differ from those for monkeys that received control MAb. Furthermore, unusual clinical sequelae of MV infection were not observed in these monkeys. In contrast, MV-infected rhesus monkeys depleted of both CD20+ and CD8+ lymphocytes had a prolonged duration of viremia and developed a desquamating skin rash. These findings indicate that humoral immunity plays a limited role in the control of MV replication in an MV-naive individual and suggest that new measles vaccination strategies should focus on the elicitation of cell-mediated immune responses, in addition to neutralizing antibodies, to facilitate rapid elimination of locally replicating virus.

Antibody-Dependent Cell-Mediated Cytotoxicity in Simian Immunodeficiency Virus-Infected Rhesus Monkeys

ABSTRACT With the recent demonstration in the RV144 Thai trial that a vaccine regimen that does not elicit neutralizing antibodies or cytotoxic T lymphocytes may confer protection against human immunodeficiency virus type 1 (HIV-1) infection, attention has turned to nonneutralizing antibodies as a possible mechanism of vaccine protection. In the current study, we evaluated the kinetics of the antibody-dependent cell-mediated cytotoxicity (ADCC) response during acute and chronic SIVmac251 infection of rhesus monkeys. We first adapted a flow cytometry-based ADCC assay, evaluating the use of different target cells as well as different strategies for quantitation of activated natural killer (NK) cells. We found that the use of SIVmac251 Env gp130-coated target cells facilitates analyses of ADCC activity with a higher degree of sensitivity than the use of simian immunodeficiency virus (SIV)-infected target cells; however, the kinetics of the measured responses were the same using these different target cells. By comparing NK cell expression of CD107a with NK cell expression of other cytokines or chemokine molecules, we found that measuring CD107a expression is sufficient for evaluating the anti-SIV function of NK cells. We also showed that ADCC responses can be detected as early as 3 weeks after SIVmac251 infection and that the magnitude of this antibody response is inversely associated with plasma viral RNA levels in animals with moderate to high levels of viral replication. However, we also demonstrated an association between NK cell-mediated ADCC responses and the amount of SIVmac251 gp140 binding antibody that developed after viral infection. This final observation raises the possibility that the antibodies that mediate ADCC are a subset of the antibodies detected in a binding assay and arise within weeks of infection.

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.

Origin and Evolution of HIV-1 in Breast Milk Determined by Single-Genome Amplification and Sequencing

ABSTRACT HIV transmission via breastfeeding accounts for a considerable proportion of infant HIV acquisition. However, the origin and evolution of the virus population in breast milk, the likely reservoir of transmitted virus variants, are not well characterized. In this study, HIV envelope (env) genes were sequenced from virus variants amplified by single-genome amplification from plasmas and milk of 12 chronically HIV-infected, lactating Malawian women. Maximum likelihood trees and statistical tests of compartmentalization revealed interspersion of plasma and milk HIV env sequences in the majority of subjects, indicating limited or no compartmentalization of milk virus variants. However, phylogenetic tree analysis further revealed monotypic virus variants that were significantly more frequent in milk (median proportion of identical viruses, 29.5%; range, 0 to 61%) than in plasma (median proportion of identical viruses, 0%; range, 0 to 26%) (P = 0.002), suggesting local virus replication in the breast milk compartment. Moreover, clonally amplified virus env genes in milk produced functional virus Envs that were all CCR5 tropic. Milk and plasma virus Envs had similar predicted phenotypes and neutralization sensitivities to broadly neutralizing antibodies in both transmitting and nontransmitting mothers. Finally, phylogenetic comparison of longitudinal milk and plasma virus env sequences revealed synchronous virus evolution and new clonal amplification of evolved virus env genes in milk. The limited compartmentalization and the clonal amplification of evolving, functional viruses in milk indicate continual seeding of the mammary gland by blood virus variants, followed by transient local replication of these variants in the breast milk compartment.