Wei Luo

Prevention of Rectal SHIV Transmission in Macaques by Daily or Intermittent Prophylaxis with Emtricitabine and Tenofovir

Background In the absence of an effective vaccine, HIV continues to spread globally, emphasizing the need for novel strategies to limit its transmission. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs could prove to be an effective intervention strategy if highly efficacious and cost-effective PrEP modalities are identified. We evaluated daily and intermittent PrEP regimens of increasing antiviral activity in a macaque model that closely resembles human transmission. Methods and Findings We used a repeat-exposure macaque model with 14 weekly rectal virus challenges. Three drug treatments were given once daily, each to a different group of six rhesus macaques. Group 1 was treated subcutaneously with a human-equivalent dose of emtricitabine (FTC), group 2 received orally the human-equivalent dosing of both FTC and tenofovir-disoproxil fumarate (TDF), and group 3 received subcutaneously a similar dosing of FTC and a higher dose of tenofovir. A fourth group of six rhesus macaques (group 4) received intermittently a PrEP regimen similar to group 3 only 2 h before and 24 h after each weekly virus challenge. Results were compared to 18 control macaques that did not receive any drug treatment. The risk of infection in macaques treated in groups 1 and 2 was 3.8- and 7.8-fold lower than in untreated macaques (p = 0.02 and p = 0.008, respectively). All six macaques in group 3 were protected. Breakthrough infections had blunted acute viremias; drug resistance was seen in two of six animals. All six animals in group 4 that received intermittent PrEP were protected. Conclusions This model suggests that single drugs for daily PrEP can be protective but a combination of antiretroviral drugs may be required to increase the level of protection. Short but potent intermittent PrEP can provide protection comparable to that of daily PrEP in this SHIV/macaque model. These findings support PrEP trials for HIV prevention in humans and identify promising PrEP modalities.

Natural Substrate Concentrations Can Modulate the Prophylactic Efficacy of Nucleotide HIV Reverse Transcriptase Inhibitors

ABSTRACT Preexposure prophylaxis (PrEP) with antiretroviral drugs is a novel human immunodeficiency virus (HIV) prevention strategy. It is generally thought that high systemic and mucosal drug levels are sufficient for protection. We investigated whether GS7340, a next-generation tenofovir (TFV) prodrug that effectively delivers tenofovir diphosphate (TFV-DP) to lymphoid cells and tissues, could protect macaques against repeated weekly rectal simian-human immunodeficiency virus (SHIV) exposures. Macaques received prophylactic GS7340 treatment 3 days prior to each virus exposure. At 3 days postdosing, TFV-DP concentrations in peripheral blood mononuclear cells (PBMCs) were about 50-fold higher than those seen with TFV disoproxil fumarate (TDF), and they remained above 1,000 fmol/106 cells for as long as 7 days. TFV-DP accumulated in lymphoid and rectal tissues, with concentrations at 3 days exceeding 500 fmol/106 mononuclear cells. Despite high mucosal and systemic TFV levels, GS7340 was not protective. Since TFV-DP blocks reverse transcription by competing with the natural dATP substrate, we measured dATP contents in peripheral lymphocytes, lymphoid tissue, and rectal mononuclear cells. Compared to those in circulating lymphocytes and lymphoid tissue, rectal lymphocytes had 100-fold higher dATP concentrations and dATP/TFV-DP ratios, likely reflecting the activated status of the cells and suggesting that TFV-DP may be less active at the rectal mucosa. Our results identify dATP/TFV-DP ratios as a possible correlate of protection by TFV and suggest that natural substrate concentrations at the mucosa will likely modulate the prophylactic efficacy of nucleotide reverse transcriptase inhibitors.

Systemic and mucosal immunological responses during repeated mucosal SHIV162P3 challenges prior to and following infection in pigtailed macaques

Local and systemic immunological changes following vaginal HIV-1 exposures are poorly characterized and may influence susceptibility to infection. Therefore, we examined longitudinal mucosal, plasma cytokine profiles and viral-specific T-cell responses (vSTRs) before and during weekly repeated low-dose SHIV(SF162P3) viral challenges in six female pigtailed macaques, even in the absence of overt systemic infection. Following a single viral challenge, induction of several cytokines was detected consistently in cervico-vaginal lavages (CVL). With additional exposure and documented systemic infection, a hallmark of response profile was defined as peak levels in both CVL (MCP-1, MIP-1alpha, TNF-alpha, IL-1beta, IL-1RA and IL-8) and plasma cytokines (MCP-1, eotaxin and IL-1RA) in the macaques. In the periphery, vSTRs were observed within the first one or two viral challenges, but prior to the detection of systemic infection in 5/6 exposed pigtailed macaques. These findings provide valuable information regarding mucosal HIV-1 infection that may benefit microbicide research and development.

Resistance to Simian HIV infection is associated with high plasma interleukin-8, RANTES and Eotaxin in a macaque model of repeated virus challenges.

Animal models for research on susceptibility to HIV are currently not available. Here we explore whether a macaque model of repeated low-dose rectal or vaginal virus challenges could be employed. We tested the hypothesis that susceptibility to Simian HIV is not merely stochastic in this model but rather is associated with identifiable host factors. Forty macaques required a median of 3.5 SHIVSF162P3 challenges for infection. We studied the association of their susceptibility with 13 predisposing plasma cytokines/chemokines (RANTES, Eotaxin, monocyte chemoattractant protein (MCP)-1, IL-7, MIP-1β, TNF-α, MIP-1α, granulocyte colony-stimulating factor, IL-8, interferon-γ, IL-17, IL-1β, IL-6). Higher plasma RANTES, IL-8, and Eotaxin were associated with lower susceptibility, that is, higher resistance to infection. In a group of macaques with low IL-8 and RANTES, a median 3 exposures were required to infect; whereas, when either IL-8 or RANTES were high, a median 12 exposures were required. Thus, susceptibility was associated with identifiable discrete host factors and was not stochastic. In addition, the macaque model identified key human resistance factors (RANTES, Eotaxin), but also revealed a novel association with resistance (IL-8). Future direct evaluation of these or other factors in the animal model may be beneficial for developing new immunomodulation strategies for HIV prevention.

Evaluation of the lymphocyte trafficking drug FTY720 in SHIVSF162P3-infected rhesus macaques

OBJECTIVES FTY720 causes retention of lymphocytes in lymphatic tissues. Previous studies revealed that FTY720 can decrease or eliminate chronic viral infections of mice. We address here whether therapeutic use of FTY720 in simian human immunodeficiency virus (SHIV)-infected rhesus macaques could also decrease viraemia. METHODS FTY720 was administered intravenously to three SHIV(SF162P3)-infected macaques at 39, 7 or 6 weeks of infection; three control macaques (47, 48 or 6 weeks of infection) did not receive drug. FTY720 was given at 0.004 mg/kg on days 0, 1, 2, 14, 15 and 16, followed by 0.1 mg/kg on days 28, 29, 30, 42, 43 and 44. Blood was collected seven times throughout and four times during 47 days of follow-up. RESULTS Only the 0.1 mg/kg dose resulted in a reduction in mean blood CD4+ T cells and B cells (to 33% and 27% of pre-drug levels, P=0.0024 and 0.003, respectively). FTY720 treatment did not lead to significant deviations from the natural pattern of viral control. Plasma viraemia progressed from a range of 10(4)-10(2) copies/mL before treatment to 10(4)-temporarily undetectable levels on the last day of treatment. SHIV(SF162P3) was not eliminated, however, as plasma viraemia and proviral DNA persisted during the follow-up. No significant alterations in T cell activity were noted throughout the drug course. CONCLUSIONS FTY720 administration had no detectable therapeutic effect at the doses and schedules outlined here, although blood CD4+ T cells and B cells were effectively reduced. Future work might reveal whether FTY720 could be beneficial in more pathogenic SHIV, simian immunodeficiency virus or HIV infections.

T Cell Chemo-Vaccination Effects after Repeated Mucosal SHIV Exposures and Oral Pre-Exposure Prophylaxis

Pre-exposure prophylaxis (PrEP) with anti-viral drugs is currently in clinical trials for the prevention of HIV infection. Induction of adaptive immune responses to virus exposures during anti-viral drug administration, i.e., a “chemo-vaccination” effect, could contribute to PrEP efficacy. To study possible chemo-vaccination, we monitored humoral and cellular immune responses in nine rhesus macaques undergoing up to 14 weekly, low-dose SHIVSF162P3 rectal exposures. Six macaques concurrently received PrEP with intermittent, oral Truvada; three were no-PrEP controls. PrEP protected 4 macaques from infection. Two of the four showed evidence of chemo-vaccination, because they developed anti-SHIV CD4+ and CD8+ T cells; SHIV-specific antibodies were not detected. Control macaques showed no anti-SHIV immune responses before infection. Chemo-vaccination-induced T cell responses were robust (up to 3,940 SFU/106 PBMCs), predominantly central memory cells, short-lived (≤22 weeks), and appeared intermittently and with changing specificities. The two chemo-vaccinated macaques were virus-challenged again after 28 weeks of rest, after T cell responses had waned. One macaque was not protected from infection. The other macaque concurrently received additional PrEP. It remained uninfected and T cell responses were boosted during the additional virus exposures. In summary, we document and characterize PrEP-induced T cell chemo-vaccination. Although not protective after subsiding in one macaque, chemo-vaccination-induced T cells warrant more comprehensive analysis during peak responses for their ability to prevent or to control infections after additional exposures. Our findings highlight the importance of monitoring these responses in clinical PrEP trials and suggest that a combination of vaccines and PrEP potentially might enhance efficacy.

Repeated Rectal SHIVSF162P3 Exposures Do Not Consistently Induce Sustained T Cell Responses Prior to Systemic Infection in the Repeat-Low Dose Preclinical Macaque Model

The macaque model of repeated SHIV exposures is increasingly used as a preclinical tool to evaluate biomedical HIV intervention strategies. It is unclear whether multiple virus exposures induce immune responses in macaques, as documented in uninfected individuals repeatedly exposed to HIV. We here address whether repeated, rectal SHIV(SF162P3) exposures lead to systemic T cell activation in 12 rhesus macaques, and whether this is associated with increased infection resistance. Eight macaques became systemically infected after 2-7 exposures, three macaques were less susceptible (infection after 10-12 exposures), and one macaque remained uninfected after 14 exposures. PBMCs were retrospectively monitored for increases in T cell activation by analyzing the proportion of CD8(+) T cells, recently activated or proliferated T cells (markers CD38, Ki67), a marker for cytotoxicity (granzyme B), or T cell-produced plasma cytokines (IFN-gamma, RANTES, IL-2). Repeated virus exposures did not induce sustained, potent, or diverse T cell responses prior to systemic infection. Some changes occurred in the analyzed parameters during repeated virus exposures, but similar T cell activities were also observed in five SHIV-unexposed control macaques. Thus, we found no evidence that delayed infection or resistance to infection was associated with systemic, long-lasting, protective T cell responses to repeated rectal virus exposures. Our results provide further insights into the repeat exposure macaque model. We find that this model can be used for testing biomedical prevention strategies without concern of eliciting a systemic vaccination effect.

Short Communication: No Evidence of Occult SHIV Infection as Demonstrated by CD8+ Cell Depletion after Chemoprophylaxis-Induced Protection from Mucosal Infection in Rhesus Macaques

Preexposure prophylaxis (PrEP) with antiretroviral drugs constitutes a promising strategy for HIV prevention. Potent PrEP regimens with reverse transcriptase inhibitors can prevent detectable SHIV infection in a repeated low-dose macaque model that resembles human transmission, supporting plans to quickly move this approach into human trials. However, the possibility remains that extremely low levels of virus replication could nonetheless occur during PrEP and seed viral reservoirs in tissues. Therefore, seemingly protected macaques may harbor occult virus that may be initially contained by cytotoxic T cells, but could emerge later. To explore this possibility, we studied whether CD8(+) cells suppress viremia in four rhesus macaques apparently protected by daily or intermittent Truvada (FTC and tenofovir) during 14 low-dose, rectal SHIV(SF162P3) challenges and during a subsequent drug washout period. CD8(+) cells were efficiently ablated with antibodies in these and two additional control macaques that were previously infected but had reached undetectable virus set points. During 4 weeks of follow-up, all four macaques remained free of plasma viremia and provirus in blood lymphocytes. In contrast, plasma viremia resurged to 10(6) to 10(7) copies per milliliter within 2 weeks in both control macaques. Thus, these results indicate that the undetectable viremia in the PrEP-protected macaques was not due to CD8(+) cells that were containing a low-level infection. Rather, the PrEP treatment created conditions in which infection was prevented, eliminated, or controlled by unknown mechanisms. These data provide important information for PrEP usage to prevent HIV transmission, and fully support the continued pursuit of PrEP prevention measures in humans.

Comparison of HIV oral fluid and plasma antibody results during early infection in a longitudinal Nigerian cohort.

BACKGROUND Oral fluid (OF) testing is a less-invasive alternative to blood-based testing for HIV. The performance of HIV OF tests has not been extensively evaluated in serially collected paired specimens from seroconverters. OBJECTIVE To compare paired OF and plasma test performance in a cohort of HIV-1 seroconverters from Nigeria. STUDY DESIGN Paired plasma and OF specimens from 14 seroconverters collected during 24 months of longitudinal follow up were included in the study. Plasma and OF were tested using Avioq HIV-1 Microelisa System, and first reactivity in plasma and OF specimens was compared. OF specimens reactive by Avioq were subsequently tested by OraSure HIV-1 Western blot. Genetic Systems HIV-1 Western blot was also performed on the corresponding plasma of the first 2 Avioq-OF positive time-points. RESULTS Of the 14 seroconverters, 5 (35.7%) had concordant results between plasma and OF for all time points tested, whereas 9 (64.3%) showed reactivity on plasma before OF specimens early in infection. The median delay between plasma and OF reactivity was 29 days (range: 0 day-20 months) (p<0.0039); the median overall delay for OF compared to RNA testing was 69.5 days. Delayed antibody response with OF was observed in both males and females regardless of viral load or HIV subtypes. CONCLUSIONS Results demonstrate decreased sensitivity of OF testing compared to blood-based testing with specimens obtained early after HIV infection. Programs that utilize OF testing in populations with increased risk of incident HIV infection should understand these limitations of OF testing.

Detailed Transmission Network Analysis of a Large Opiate-Driven Outbreak of HIV Infection in the United States

In January 2015, an outbreak of undiagnosed human immunodeficiency virus (HIV) infections among persons who inject drugs (PWID) was recognized in rural Indiana. By September 2016, 205 persons in this community of approximately 4400 had received a diagnosis of HIV infection. We report results of new approaches to analyzing epidemiologic and laboratory data to understand transmission during this outbreak. HIV genetic distances were calculated using the polymerase region. Networks were generated using data about reported high-risk contacts, viral genetic similarity, and their most parsimonious combinations. Sample collection dates and recency assay results were used to infer dates of infection. Epidemiologic and laboratory data each generated large and dense networks. Integration of these data revealed subgroups with epidemiologic and genetic commonalities, one of which appeared to contain the earliest infections. Predicted infection dates suggest that transmission began in 2011, underwent explosive growth in mid-2014, and slowed after the declaration of a public health emergency. Results from this phylodynamic analysis suggest that the majority of infections had likely already occurred when the investigation began and that early transmission may have been associated with sexual activity and injection drug use. Early and sustained efforts are needed to detect infections and prevent or interrupt rapid transmission within networks of uninfected PWID.