Mars Stone

A Limited Number of Simian Immunodeficiency Virus (SIV) env Variants Are Transmitted to Rhesus Macaques Vaginally Inoculated with SIVmac251

ABSTRACT Single-genome amplification (SGA) and sequencing of HIV-1 RNA in plasma of acutely infected humans allows the identification and enumeration of transmitted/founder viruses responsible for productive systemic infection. Use of this strategy as a means for identifying transmitted viruses suggested that intrarectal simian immunodeficiency virus (SIV) inoculation of macaques recapitulates key features of human rectal infection. However, no studies have used the SGA strategy to identify vaginally transmitted virus(es) in macaques or to determine how early SIV diversification in vaginally infected animals compares with HIV-1 in humans. We used SGA to amplify 227 partial env sequences from a SIVmac251 challenge stock and from seven rhesus macaques at the earliest plasma viral RNA-positive time point after low- and high-dose intravaginal inoculation. Sequences were analyzed phylogenetically to determine the relationship of transmitted/founder viruses within and between each animal and the challenge stock. In each animal, discrete low-diversity env sequence lineages were evident, and these coalesced phylogenetically to identical or near-identical env sequences in the challenge stock, thus confirming the validity of the SGA sequencing and modeling strategy for identifying vaginally transmitted SIV. Between 1 and 10 viruses were responsible for systemic infection, similar to humans infected by sexual contact, and the set of viruses transmitted to the seven animals studied represented the full genetic constellation of the challenge stock. These findings recapitulate many of the features of sexual HIV-1 transmission in women. Furthermore, the SIV rhesus macaque model can be used to understand the factors that influence the transmission of single versus multiple SIV variants.

High Specific Infectivity of Plasma Virus from the Pre-Ramp-Up and Ramp-Up Stages of Acute Simian Immunodeficiency Virus Infection

ABSTRACT To define the ratio of simian immunodeficiency virus (SIV) RNA molecules to infectious virions in plasma, a ramp-up-stage plasma pool was made from the earliest viral RNA (vRNA)-positive plasma samples (collected approximately 7 days after inoculation) from seven macaques, and a set-point-stage plasma pool was made from plasma samples collected 10 to 16 weeks after peak viremia from seven macaques; vRNA levels in these plasma pools were determined, and serial 10-fold dilutions containing 1 to 1,500 vRNA copies/ml were made. Intravenous (i.v.) inoculation of a 1-ml aliquot of diluted ramp-up-stage plasma containing 20 vRNA copies infected 2 of 2 rhesus macaques, while for the set-point-stage plasma, i.v. inoculation with 1,500 vRNA copies was needed to transmit infection. Further, when the heat-inactivated set-point-stage plasma pool was mixed with ramp-up-stage virions, infection of inoculated macaques was blocked. Notably, 2 of 2 animals inoculated with 85 ml of a pre-ramp-up plasma pool containing <3 SIV RNA copies/ml developed SIV infections characterized by high levels of viral replication, demonstrating that “vRNA-negative” plasma collected from macaques in the pre-ramp-up stage is infectious. Furthermore, there is a high ratio of infectious virions to total virions in ramp-up-stage plasma (between 1:1 and 1:10) and a lower ratio in set-point-stage plasma (between 1:75 and 1:750). Heat-inactivated chronic-stage plasma can “neutralize” the highly infectious ramp-up-stage virions. These findings have implications for the understanding of the natural history of SIV and human immunodeficiency virus infection and transmission.

SIVmac251 Is Inefficiently Transmitted to Rhesus Macaques by Penile Inoculation with a Single SIVenv Variant Found in Ramp-up Phase Plasma

Abstract Despite the fact that approximately half of all HIV patients acquire infection through penile exposure, there have been no recent studies of penile SIV transmission in rhesus macaques and the nature of the virus variants transmitted, target cells, and pathways of virus dissemination to systemic lymphoid tissues are not known. Single genome amplification (SGA) and sequencing of HIV-1 RNA in plasma of acutely infected humans allows the identification and enumeration of transmitted/founder viruses responsible for productive systemic infection. Studies using the SGA strategy have shown that intrarectal and intravaginal SIV transmission to macaques recapitulates key features of human HIV transmission. To date, no studies have used the SGA assay to identify transmitted/founder virus(es) in macaques infected after penile SIV exposure. Here we report that SIV can be transmitted by penile SIV exposure. However, similar exposure to a high-dose inoculum infects only about half the animals, which is about 50% less efficient transmission than occurs after vaginal SIV challenge. In addition, only a single SIV env variant established the systemic infection in all five animals that became infected after penile exposure, a result that is consistent with low incidence and few transmitted HIV variants in heterosexually infected men. Our results suggest that the penile transmission of SIVmac251 in rhesus macaques recapitulates the key features of penile HIV-1 transmission and may provide insight into host or viral factors that permit penile transmission and dissemination. Furthermore, this SIV challenge exposure route will be useful in testing vaccines and other prophylactic approaches.

Zika Virus Tissue and Blood Compartmentalization in Acute Infection of Rhesus Macaques.

Animal models of Zika virus (ZIKV) are needed to better understand tropism and pathogenesis and to test candidate vaccines and therapies to curtail the pandemic. Humans and rhesus macaques possess similar fetal development and placental biology that is not shared between humans and rodents. We inoculated 2 non-pregnant rhesus macaques with a 2015 Brazilian ZIKV strain. Consistent with most human infections, the animals experienced no clinical disease but developed short-lived plasma viremias that cleared as neutralizing antibody developed. In 1 animal, viral RNA (vRNA) could be detected longer in whole blood than in plasma. Despite no major histopathologic changes, many adult tissues contained vRNA 14 days post-infection with highest levels in hemolymphatic tissues. These observations warrant further studies to investigate ZIKV persistence and its potential clinical implications for transmission via blood products or tissue and organ transplants.

First Zika-positive donations in the continental United States

Zika virus (ZIKV) has spread in the Americas, including parts of the southern United States, and infection can be associated with serious complications, including congenital brain abnormalities. Probable transfusion transmission of ZIKV has been documented in Brazil.

Limited dissemination of pathogenic SIV after vaginal challenge of rhesus monkeys immunized with a live, attenuated lentivirus.

In non-human primate models of AIDS, attenuated lentiviruses provide the most reliable protection from challenge with pathogenic virus but the extent to which the vaccine virus replicates after challenge is unclear. At 7 and 14 days after vaginal challenge with pathogenic SIVmac239, plasma SIVenv RNA levels were significantly lower in female macaques immunized 6 months earlier with live, attenuated SHIV89.6 compared to unimmunized control animals. In 2 SHIV-immunized, unprotected macaques SIV replication produced moderate-level plasma viremia with dissemination of challenge virus to all tissues on day 14 after challenge. In protected, SHIV-immunized monkeys, SIV replication was controlled in all tissues, from the day of challenge through 14 days post-challenge. Further, in CD8(+) T cell-depleted SHIV-immunized animals, SIV replication and dissemination were more rapid than in control animals. These findings suggest that replication of a pathogenic AIDS virus can be controlled at the site of mucosal inoculation by live-attenuated lentivirus immunization.

Prior Dengue Virus Exposure Shapes T Cell Immunity to Zika Virus in Humans

ABSTRACT While progress has been made in characterizing humoral immunity to Zika virus (ZIKV) in humans, little is known regarding the corresponding T cell responses to ZIKV. Here, we investigate the kinetics and viral epitopes targeted by T cells responding to ZIKV and address the critical question of whether preexisting dengue virus (DENV) T cell immunity modulates these responses. We find that memory T cell responses elicited by prior infection with DENV or vaccination with tetravalent dengue attenuated vaccines (TDLAV) recognize ZIKV-derived peptides. This cross-reactivity is explained by the sequence similarity of the two viruses, as the ZIKV peptides recognized by DENV-elicited memory T cells are identical or highly conserved in DENV and ZIKV. DENV exposure prior to ZIKV infection also influences the timing and magnitude of the T cell response. ZIKV-reactive T cells in the acute phase of infection are detected earlier and in greater magnitude in DENV-immune patients. Conversely, the frequency of ZIKV-reactive T cells continues to rise in the convalescent phase in DENV-naive donors but declines in DENV-preexposed donors, compatible with more efficient control of ZIKV replication and/or clearance of ZIKV antigen. The quality of responses is also influenced by previous DENV exposure, and ZIKV-specific CD8 T cells from DENV-preexposed donors selectively upregulated granzyme B and PD1, unlike DENV-naive donors. Finally, we discovered that ZIKV structural proteins (E, prM, and C) are major targets of both the CD4 and CD8 T cell responses, whereas DENV T cell epitopes are found primarily in nonstructural proteins. IMPORTANCE The issue of potential ZIKV and DENV cross-reactivity and how preexisting DENV T cell immunity modulates Zika T cell responses is of great relevance, as the two viruses often cocirculate and Zika virus has been spreading in geographical regions where DENV is endemic or hyperendemic. Our data show that memory T cell responses elicited by prior infection with DENV recognize ZIKV-derived peptides and that DENV exposure prior to ZIKV infection influences the timing, magnitude, and quality of the T cell response. Additionally, we show that ZIKV-specific responses target different proteins than DENV-specific responses, pointing toward important implications for vaccine design against this global threat.

Development of a contemporary globally diverse HIV viral panel by the EQAPOL program

The significant diversity among HIV-1 variants poses serious challenges for vaccine development and for developing sensitive assays for screening, surveillance, diagnosis, and clinical management. Recognizing a need to develop a panel of HIV representing the current genetic and geographic diversity NIH/NIAID contracted the External Quality Assurance Program Oversight Laboratory (EQAPOL) to isolate, characterize and establish panels of HIV-1 strains representing global diverse subtypes and circulating recombinant forms (CRFs), and to make them available to the research community. HIV-positive plasma specimens and previously established isolates were collected through a variety of collaborations with a preference for samples from acutely/recently infected persons. Source specimens were cultured to high-titer/high-volume using well-characterized cryopreserved PBMCs from National y donors. Panel samples were stored as neat culture supernatant or diluted into defibrinated plasma. Characterization for the final expanded virus stocks included viral load, p24 antigen, infectivity (TCID), sterility, coreceptor usage, and near full-length genome sequencing. Viruses are made available to approved, interested laboratories using an online ordering application. The current EQAPOL Viral Diversity panel includes 100 viral specimens representing 6 subtypes (A, B, C, D, F, and G), 2 sub-subtypes (F1 and F2), 7 CRFs (01, 02, 04, 14, 22, 24, and 47), 19 URFs and 3 group O viruses from 22 countries. The EQAPOL Viral Diversity panel is an invaluable collection of well-characterized reagents that are available to the scientific community, including researchers, epidemiologists, and commercial manufacturers of diagnostics and pharmaceuticals to support HIV research, as well as diagnostic and vaccine development.

Relative analytical sensitivity of donor nucleic acid amplification technology screening and diagnostic real-time polymerase chain reaction assays for detection of Zika virus RNA

Zika virus (ZIKV) has spread rapidly in the Pacific and throughout the Americas and is associated with severe congenital and adult neurologic outcomes. Nucleic acid amplification technology (NAT) assays were developed for diagnostic applications and for blood donor screening on high‐throughput NAT systems. We distributed blinded panels to compare the analytical performance of blood screening relative to diagnostic NAT assays.

First cases of Zika virus–infected US blood donors outside states with areas of active transmission

Zika virus (ZIKV) is transmitted by Aedes mosquitos and can result in severe congenital and adult neurologic abnormalities. ZIKV has rapidly spread northward through Central America and the Caribbean and autochthonous cases have been identified in the continental United States. High rates of ZIKA RNA positivity were detected in blood donors during previous epidemics. ZIKV transmission by transfused blood from healthy donor components has been a growing concern.