Silvina Masciotra

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.

Intermittent Prophylaxis with Oral Truvada Protects Macaques from Rectal SHIV Infection

Treating monkeys with single doses of an antiretroviral drug before and after exposure to SHIV provides protection against infection, a schedule that may prove practical in humans. Rearranging Retroviral Regimens for HIV Antiretroviral drugs have transformed the lives of HIV-infected people by preventing progression to full-blown AIDS. These drugs also dramatically reduce HIV transmission from mothers to infants during pregnancy and breastfeeding, and work in monkeys suggests that daily doses can also reduce transmission from unprotected sex. But prophylactic treatment with antiretroviral drugs is costly and impractical—even if confined to a high-risk population. García-Lerma et al. now show that in monkeys a more realistic medication schedule may work just as well as daily doses. To simulate how people are likely to be infected with HIV, the authors exposed macaque monkeys rectally to 14 weekly doses of simian-human immunodeficiency virus (SHIV) engineered to resemble the human virus. Control macaques treated in this way became infected within the first five exposures to SHIV. Researchers then assessed whether oral, human-equivalent doses of antiretroviral agents could prevent infection in monkeys. The best protection—equivalent to that provided by daily antivirals—occurred when the drug Truvada was given 1, 3, or 7 days before virus exposure followed by a second dose 2 hours after exposure. Less effective, but still better than no treatment at all, was a schedule in which the drug was given 2 hours before or after exposure and then again 24 hours later. Drugs given only 24 or 48 hours after exposure did not safeguard against infection. The results of this study are preliminary, largely because each of the groups had only six macaques, but they are nevertheless promising. If ongoing clinical trials in healthy people show that daily antiretroviral therapy can diminish the chances of acquiring HIV after exposure, a reasonable next step would be to evaluate more practical, less costly drug schedules in humans. For example, a weekly dose followed by a second dose after a possible exposure could prove both effective and tractable. It will also be important to evaluate treatments based solely on exposure, as these would not require ongoing prophylactic drug treatment and would minimize any drug toxicity. If one or more of these therapeutic regimens is successful, antiretroviral drugs may expand the transformation they have already engendered by preventing many more new infections as well as controlling existing ones. HIV continues to spread globally, mainly through sexual contact. Despite advances in treatment and care, preventing transmission with vaccines or microbicides has proven difficult. A promising strategy to avoid transmission is prophylactic treatment with antiretroviral drugs before exposure to HIV. Clinical trials evaluating the efficacy of daily treatment with the reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) or Truvada (TDF plus emtricitabine) are under way. We hypothesized that intermittent prophylactic treatment with long-acting antiviral drugs would be as effective as daily dosing in blocking the earliest stages of viral replication and preventing mucosal transmission. We tested this hypothesis by intermittently giving prophylactic Truvada to macaque monkeys and then exposing them rectally to simian-human immunodeficiency virus (SHIV) once a week for 14 weeks. A simple regimen with an oral dose of Truvada given 1, 3, or 7 days before exposure followed by a second dose 2 hours after exposure was as protective as daily drug administration, possibly because of the long intracellular persistence of the drugs. In addition, a two-dose regimen initiated 2 hours before or after virus exposure was effective, and full protection was obtained by doubling the Truvada concentration in both doses. We saw no protection if the first dose was delayed until 24 hours after exposure, underscoring the importance of blocking initial replication in the mucosa. Our results show that intermittent prophylactic treatment with an antiviral drug can be highly effective in preventing SHIV infection, with a wide window of protection. They strengthen the possibility of developing feasible, cost-effective strategies to prevent HIV transmission in humans.

Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infections

BACKGROUND The current algorithm for HIV diagnosis in the US involves screening with an immunoassay (IA) and supplemental testing with Western blot (WB) or immunofluorescence assay. Because of existence of more sensitive and specific FDA-approved assays that would also reduce the cost and turn-around time of testing compared to WB, several alternative algorithms have been evaluated. Recently, an alternative algorithm using a sensitive 3rd or 4th generation IA followed by an HIV-1 and HIV-2 discriminatory supplemental test on the initial IA-positive specimens was proposed. Concordant positive results indicate HIV-positive specimens and discordant results are resolved by nucleic acid amplification testing (NAAT). OBJECTIVES To evaluate the sensitivity of assays during acute HIV infection and the performance of the current and an alternative algorithm using samples from HIV-1 seroconversion panels and persons with established HIV infections. STUDY DESIGN To evaluate the algorithms in early infections, 26 HIV-1 seroconverters from the US were tested with three 3rd generation and one 4th generation IA, six rapid tests (RTs), one NAAT, and WB. Sensitivity and specificity of the algorithms were calculated by testing an additional 416 HIV-positive and 414 uninfected control samples with one 3rd generation and one 4th generation IA, four RTs, one NAAT, and WB. RESULTS The individual assays evaluated became positive 5 (RT) to 26 days (NAAT) before WB was positive. Among seroconverters, the alternative algorithm detected significantly more infections than the current algorithm (103-134 versus 56, p<0.0001). Furthermore, the use of a 4th generation IA instead of a 3rd generation assay as the screen resulted in significantly higher detection of acute infections (p<0.0001). In contrast, the algorithms performed equally among specimens from established HIV-1 infections. CONCLUSIONS This study demonstrated improved sensitivity of the alternative algorithm for detecting acute HIV-1 infections, while maintaining the ability to accurately detect established HIV-1 infections. Early detection is important as individuals can be highly infectious during acute infection. In addition, the alternative algorithm should reduce turn-around time by using a RT as the supplemental test has the potential to increase the number of test results returned.

High concordance between HIV-1 drug resistance genotypes generated from plasma and dried blood spots in antiretroviral-experienced patients.

Objective:Dried blood spots (DBS) are a convenient alternative to plasma for drug resistance testing in resource-limited settings. We investigated the correlation between resistance genotypes generated from DBS and plasma. Design:Sixty DBS specimens from HIV-1 subtype B-infected antiretroviral-experienced (n = 58) and naive patients (n = 2) were tested. DBS were prepared using 50 μl blood and were stored with desiccant at −20°C. Methods:Resistance genotypes from DBS were obtained using the ViroSeq HIV-1 assay and were compared with genotypes derived from plasma. The frequency of amplification of proviral DNA from DBS was evaluated using an in-house nested polymerase chain reaction assay. Results:Fifty of the 60 DBS specimens were successfully genotyped including all 38 specimens collected from patients with plasma viral loads greater than 2000 copies/ml and 12 of 22 DBS (54.5%) from patients with viral loads less than 2000 copies/ml. HIV-1 DNA was detected in 44.4% of the DBS. Despite the presence of DNA, genotypes from DBS and plasma were highly concordant. Of the 316 mutations found in plasma sequences, 306 (96.8%) were also found in DBS. Discrepancies were mostly caused by mixtures at minor protease positions or unusual amino acid changes, and in only two cases were caused by major protease (M46L) or reverse transcriptase (K103N) mutations absent in DBS sequences. Conclusion:We demonstrated a high concordance between resistance genotypes from plasma and DBS, and that resistance testing from DBS can achieve sensitive levels similar to those seen using plasma. Our results indicate that DBS may represent a feasible alternative to plasma for drug resistance testing in treated individuals.

HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4°C

Background Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at −20°C for prolonged periods (0.5–4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4°C for 1 year. Methods A total of 40 DBSs were prepared from residual diagnostic specimens collected from HIV subtype B-infected persons and were stored with desiccant at 4°C. Total nucleic acids were extracted after 1 year using a modification of the Nuclisens assay. Resistance testing was performed using the ViroSeq HIV-1 assay and an in-house nested RT–PCR method validated for HIV-1 subtype B that amplifies a smaller (1 kb) pol fragment. Results Using the ViroSeq assay, only 23 of the 40 (57.5%) DBS specimens were successfully genotyped; 22 of these specimens had plasma viraemia >10 000 RNA copies/mL. When the specimens were tested using the in-house assay, 38 of the 40 DBSs (95%) were successfully genotyped. Overall, resistance genotypes generated from the DBSs and plasma were highly concordant. Conclusions We show that drug resistance genotyping from DBSs stored at 4°C with desiccant is highly efficient but requires the amplification of small pol fragments and the use of an in-house nested PCR protocol with quality-controlled reagents. These findings suggest that 4°C may represent a suitable temperature for long-term storage of DBSs.

Simian Immunodeficiency Viruses of Diverse Origin Can Use CXCR4 as a Coreceptor for Entry into Human Cells

ABSTRACT Primary simian immunodeficiency virus (SIV) isolated from sooty mangabey (SIVsm [n = 6]), stumptail (SIVstm [n = 1]), mandrill (SIVmnd [n = 1]), and African green (SIVagm [n = 1]) primates were examined for their ability to infect human cells and for their coreceptor requirements. All isolates infected human peripheral blood mononuclear cells (PBMCs) from a CCR5+/+ donor, and seven of eight isolates tested also infected CCR5−/− PBMCs. Analysis of coreceptor utilization using GHOST and U87 cell lines revealed that all of the isolates tested used CCR5 and the orphan receptors STRL33 and GPR15. Coreceptors such as CCR2b, CCR3, CCR8, and CX3CR1 were also utilized by some primary SIV isolates. More importantly, we found that CXCR4 was used as a coreceptor by the SIVstm, the SIVagm, and four of the SIVsm isolates in GHOST and U87 cells. These data suggest that primary SIV isolates from diverse primate species can utilize CXCR4 for viral entry, similar to what has been described for human immunodeficiency viruses.

Temporal relationship between V1V2 variation, macrophage replication, and coreceptor adaptation during HIV-1 disease progression

Background: Specific mutations in VPR and V2 potentially restrict HIV-1 replication in macrophages. Such restriction could potentially limit HIV replication in long-term non-progressors (LTNP), thus accounting for low viral load and delayed progression to AIDS. Objective: To examine whether a specific VPR phenotype (truncated versus non-truncated) correlates with disease progression and whether elongated V2 restricts viral replication in macrophages or alters viral tropism. Methods: Sequence analysis was carried for VPR and V1-V3 env from four rapid progressors (RPs), six late progressors (LPs), and three LTNPs in cohort of HIV-1-infected homosexual men. The replication kinetics of sequential isolates was examined in primary CD4 cells and macrophages and coreceptor usage was determined by GHOST infection assays. Results: No differences were found in the VPR protein from RP and LTNP isolates. Analysis of the V2 region revealed that all RPs maintained similar V2 lengths (40 aa), whereas LPs and LTNPs acquired additional amino acids (2-13 aa) in the V2 region. Coreceptor specificity revealed that RP switch from CCR5 to multiple coreceptor usage, whereas LTNPs maintained R5 viruses. Sequential isolates from each group revealed comparable replication efficiencies in both T-cells and macrophages, regardless of the V2 length or coreceptor utilization. In addition, cross-section analysis of six LTNPs from Australia revealed extended V2 with consistent usage of CCR5 coreceptor. Conclusion: The present results suggest that acquisition of a V2 extension over time in HIV-1-infected LPs/LTNPs appears to correlate with maintenance of CCR5 usage among LTNPs. These findings may be important for a better understanding of the host interactions and disease progression.

Performance of a Fourth-Generation HIV Screening Assay and an Alternative HIV Diagnostic Testing Algorithm

Objective:We evaluated the performance of the GS fourth-generation antigen/antibody assay and compared Centers for Disease Control and Preventions (CDCs) proposed alternative algorithm [repeatedly reactive fourth-generation immunoassay followed by an HIV-1/HIV-2 differentiation immunoassay and, if needed, nucleic acid test (NAT)] with the current algorithm (repeatedly reactive third-generation immunoassay followed by HIV-1 western blot). Design:A convenience sample of the following four specimen sets was acquired: 10 014 from insurance applicants, 493 known western blot-positive, 20 known western blot-indeterminate specimens, and 230 specimens from 26 HIV-1 seroconverters. Methods:Specimens were tested with the GS third-generation and fourth-generation immunoassays, the Multispot HIV-1/HIV-2 differentiation immunoassay, NAT, and western blot. We applied the two algorithms using these results. Results:Among insurance specimens, 13 (0.13%) specimens were immunoassay repeatedly reactive: two were HIV-positive (repeatedly reactive by third-generation and fourth-generation immunoassays, and western blot and Multispot positive); two third-generation repeatedly reactive and nine fourth-generation repeatedly reactive specimens were false-positive. Third-generation and fourth-generation specificities were 99.98% [95% confidence interval (CI) 99.93–100%] and 99.91% (95% CI 99.84–99.96%), respectively.All HIV-1 western blot-positive specimens were repeatedly reactive by third-generation and fourth-generation immunoassays. By Multispot, 491 (99.6%) were HIV-1-positive and two (0.4%) were HIV-2-positive.Only eight (40%) western blot-indeterminate specimens were fourth-generation repeatedly reactive: six were Multispot and NAT-negative and two were Multispot HIV-1-positive but NAT-negative.The alternative algorithm correctly classified as positive 102 seroconverter specimens with the third-generation immunoassay and 130 with the fourth-generation immunoassay compared with 56 using the western blot with either immunoassay. Conclusion:The alternative testing algorithm improved early infection sensitivity and identified HIV-2 infections. Two potential false-positive algorithm results occurred with western blot-indeterminate specimens.

Sequence Note: Evidence of a High Frequency of HIV-1 Subtype F Infections in a Heterosexual Population in Buenos Aires, Argentina

We analyzed HIV-1 genetic variability, phylogenetic relationships, and association with transmission modes among 58 HIV-1-infected patients from Buenos Aires City, Argentina. The 58 strains were classified as envgp41 HIV-1 group M subtype B (n = 34) and subgroup F1 of subtype F (n = 24). Potential recombinants combining parts of viral regions from different subtypes, Bprot/Fenv and Fprot/Benv, were found in two patients, and a dual infection with HIV-1 prot subtypes B and F was identified in one individual. Epidemiologic analysis of behavioral risks revealed that the frequency of infection with subtype F viruses was significantly higher (p < 0.0001) among heterosexual patients (71%) compared with homosexual patients (11%). The spread of non-B subtypes into heterosexual populations may be more common than previously thought. Our findings provide important information for monitoring the transmission of HIV-1 strains among different risk groups in Argentina as well as for vaccine development.

Evaluation of United States-Licensed Human Immunodeficiency Virus Immunoassays for Detection of Group M Viral Variants

ABSTRACT Six Food and Drug Administration (FDA)-licensed human immunodeficiency virus type 1 (HIV-1) and HIV-1/2 immunoassays, including five enzyme immunoassays and one rapid test, were challenged with up to 250 serum samples collected from various global sites. The serum samples were from individuals known to be infected with variants of HIV-1 including group M subtypes A, B, B′, C, D, E, F, and G and group O. All immunoassays detected the vast majority of samples tested. Three samples produced low signal over cutoff values in one or more tests: a clade B sample, an untypeable sample with a low antibody titer, and a group O sample. It is concluded that HIV-1 immunoassays used in the United States are capable of detecting most HIV-1 group M variants.