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1.  Emergence of CD4 Independence Envelopes and Astrocyte Infection in R5 Simian-Human Immunodeficiency Virus Model of Encephalitis 
Journal of Virology  2014;88(15):8407-8420.
ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) infection in the central nervous system (CNS) is characterized by replication in macrophages or brain microglia that express low levels of the CD4 receptor and is the cause of HIV-associated dementia and related cognitive and motor disorders that affect 20 to 30% of treatment-naive patients with AIDS. Independent viral envelope evolution in the brain has been reported, with the need for robust replication in resident CD4low cells, as well as CD4-negative cells, such as astrocytes, proposed as a major selective pressure. We previously reported giant-cell encephalitis in subtype B and C R5 simian-human immunodeficiency virus (SHIV)-infected macaques (SHIV-induced encephalitis [SHIVE]) that experienced very high chronic viral loads and progressed rapidly to AIDS, with varying degrees of macrophage or microglia infection and activation of these immune cells, as well as astrocytes, in the CNS. In this study, we characterized envelopes (Env) amplified from the brains of subtype B and C R5 SHIVE macaques. We obtained data in support of an association between severe neuropathological changes, robust macrophage and microglia infection, and evolution to CD4 independence. Moreover, the degree of Env CD4 independence appeared to correlate with the extent of astrocyte infection in vivo. These findings further our knowledge of the CNS viral population phenotypes that are associated with the severity of HIV/SHIV-induced neurological injury and improve our understanding of the mechanism of HIV-1 cellular tropism and persistence in the brain.
IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) infection of astrocytes in the brain has been suggested to be important in HIV persistence and neuropathogenesis but has not been definitively demonstrated in an animal model of HIV-induced encephalitis (HIVE). Here, we describe a new nonhuman primate (NHP) model of R5 simian-human immunodeficiency virus (SHIV)-induced encephalitis (SHIVE) with several classical HIVE features that include astrocyte infection. We further show an association between severe neuropathological changes, robust resident microglia infection, and evolution to CD4 independence of viruses in the central nervous system (CNS), with expansion to infection of truly CD4-negative cells in vivo. These findings support the use of the R5 SHIVE models to study the contribution of the HIV envelope and viral clades to neurovirulence and residual virus replication in the CNS, providing information that should guide efforts to eradicate HIV from the body.
doi:10.1128/JVI.01237-14
PMCID: PMC4135954  PMID: 24829360
2.  HSV-2-Driven Increase in the Expression of α4β7 Correlates with Increased Susceptibility to Vaginal SHIVSF162P3 Infection 
PLoS Pathogens  2014;10(12):e1004567.
The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of α4β7 is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of α4β7 and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIVSF162P3 infection, had higher frequency of α4β7high CD4+ T cells in the vaginal tissue and higher expression of α4β7 and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIVSF162P3. HSV-2 infection increased the frequencies of α4β7high CD4+ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of α4β7high CD4+ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4+ T cells and DCs that express high levels of α4β7 is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of α4β7 positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission.
Author Summary
Understanding the factors that correlate with an increased risk of acquiring HIV infection is key to identify new means of preventing HIV transmission. HSV-2 infection increases the risk of HIV transmission even in absence of visible lesions and inflammation. In order to explore HSV-2− associated factors that could explain this phenomenon, we used a model of asymptomatic HSV-2 infection in macaques and ex vivo cultures of biopsied vaginal tissue. We determined that HSV-2 infection is associated with an increase in subsets of immune cells that express high levels of α4β7, a molecule needed by the cells to reach the gut and the gut lymphoid tissues. The gut is an important site for HIV infection and pathogenesis and CD4+ T cells expressing high levels of α4β7 (α4β7high) are highly susceptible to the virus. We determined that the HSV-2-driven increase in these cells correlates with an increased susceptibility of the vaginal mucosa to SIV infection. Thus, our results suggest that an increased availability of α4β7high cells at the mucosal site of HIV exposure may constitute a risk factor for HIV acquisition in HSV-2 positive and, possibly, negative individuals.
doi:10.1371/journal.ppat.1004567
PMCID: PMC4270786  PMID: 25521298
3.  The frequency of α4β7high memory CD4+ T cells correlates with susceptibility to rectal SIV infection 
Journal of acquired immune deficiency syndromes (1999)  2013;64(4):10.1097/QAI.0b013e31829f6e1a.
Background
Integrin α4β7 (α4β7) mediates the homing of CD4+ T cells to gut-associated lymphoid tissues (GALT), which constitute a highly favorable environment for HIV expansion and dissemination. HIV and SIV envelope proteins bind to and signal through α4β7 and during acute infection SIV preferentially infects α4β7high CD4+ T cells. We postulated that the availability of these cells at the time of challenge could influence mucosal SIV transmission and acute viral load (VL).
Methods
We challenged 17 rhesus macaques with 3000 TCID50 of SIVmac239 rectally and followed the subsets of α4β7+ T and dendritic cells (DCs) by flow cytometry in blood and tissues, before and after challenge.
Results
We found that the frequency of memory CD4+ T cells that expressed high levels of α4β7 (α4β7high memory CD4+ T cells) in blood before challenge correlated strongly with susceptibility to infection and acute VL. Notably, not only at the time of challenge, but also their frequency 3 weeks before challenge correlated with infection. This association extended to the rectal tissue as we observed a strong direct correlation between the frequency of α4β7high memory CD4+ T cells in blood and rectum before and after challenge. The frequency of α4β7+ myeloid DCs and α4β7high CD80+ DCs also correlated with infection and acute VL, while blood CCR5+ and CD69+ CD4+ T cells could not be associated with infection.
Conclusions
Our results suggest that animals with higher frequency of α4β7high CD4+ T cells in circulation and in rectal tissue could be more susceptible to SIV rectal transmission.
doi:10.1097/QAI.0b013e31829f6e1a
PMCID: PMC3815485  PMID: 23797688
HIV; SIV; mucosa transmission; integrin alpha-4 beta-7; susceptibility; gut
4.  Liposome Encapsulation of Ciprofloxacin Improves Protection against Highly Virulent Francisella tularensis Strain Schu S4 
Liposome-encapsulated ciprofloxacin for inhalation (CFI) was investigated as a putative postexposure therapeutic for two strains of Francisella tularensis. The efficacies of oral ciprofloxacin and intranasally instilled CFI could not be distinguished in a mouse model of infection with the F. tularensis live vaccine strain (LVS), where a single dose of either formulation offered full protection against a lethal challenge. However, mouse studies with the more virulent Schu S4 strain of F. tularensis demonstrated that a higher level of protection against a lethal aerosol infection is provided by CFI than by oral ciprofloxacin. In addition, using this infection model, it was possible to discriminate the efficacy of intranasally instilled CFI from that of aerosolized CFI, with aerosolized CFI providing full protection after just a single dose. The improved efficacy of CFI compared to oral ciprofloxacin is likely due to the high sustained concentrations of ciprofloxacin in the lung. In summary, CFI may be a promising therapy, perhaps enabling the prophylactic regimen to be shortened, for use in the event of a deliberate release of F. tularensis. The prophylactic efficacy of CFI against other biological warfare (BW) threat agents also warrants investigation.
doi:10.1128/AAC.02555-13
PMCID: PMC4068435  PMID: 24637682
5.  Heterogeneity in Risk of Pelvic Inflammatory Diseases After Chlamydia Infection: A Population-Based Study in Manitoba, Canada 
The Journal of Infectious Diseases  2014;210(Suppl 2):S549-S555.
Background. The association between chlamydia infection and pelvic inflammatory disease (PID) is a key parameter for models evaluating the impact of chlamydia control programs. We quantified this association using a retrospective population-based cohort.
Methods. We used administrative health data sets to construct a retrospective population-based cohort of women and girls aged 12–24 years who were resident in Manitoba, Canada, between 1992 and 1996. We performed survival analysis on a subcohort of individuals who were tested for chlamydia to estimate the risk of PID diagnosed in a primary care, outpatient, or inpatient setting after ≥1 positive chlamydia test.
Results. A total of 73 883 individuals contributed 625 621 person years of follow-up. Those with a diagnosis of chlamydia had an increased risk of PID over their reproductive lifetime compared with those who tested negative (adjusted hazard ratio [AHR], 1.55; 95% confidence interval [CI], 1.43–1.70). This risk increased with each subsequent infection: the AHR was 1.17 for first reinfection (95% CI, 1.06–1.30) and 1.35 for the second (95% CI, 1.04–1.75). The increased risk of PID from reinfection was highest in younger individuals (AHR, 4.55 (95% CI, 3.59–5.78) in individuals aged 12–15 years at the time of their second reinfection, compared with individuals older than 30 years).
Conclusions. There is heterogeneity in the risk of PID after a chlamydia infection. Describing the progression to PID in mathematical models as an average rate may be an oversimplification; more accurate estimates of the cost-effectiveness of screening may be obtained by using an individual-based measure of risk. Health inequalities may be reduced by targeting health promotion interventions at sexually active girls younger than 16 years and those with a history of chlamydia.
doi:10.1093/infdis/jiu483
PMCID: PMC4231643  PMID: 25381374
Chlamydia trachomatis; cohort study; pelvic inflammatory disease; retrospective study; epidemiology; mathematical models; cost effectiveness
6.  MIV-150-Containing Intravaginal Rings Protect Macaque Vaginal Explants against SHIV-RT Infection 
Recent studies demonstrated that intravaginal rings (IVRs) containing 100 mg of the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 significantly protect macaques against a chimeric simian-human immunodeficiency virus that expresses the HIV-1 HxB2 reverse transcriptase (SHIV-RT) when present before and after vaginal challenge. The objectives of this study were to (i) evaluate the pharmacodynamics (PD) of MIV-150 in vaginal fluids (VF) and in ectocervical and vaginal tissues following 100-mg MIV-150 IVR exposure and to (ii) gain more insight whether pharmacokinetics (PK) of MIV-150 can predict PD. MIV-150 in VF collected at 1 day and 14 days post-MIV-150 IVR insertion inhibited ex vivo SHIV-RT infection in vaginal biopsy specimens from untreated animals (not carrying IVRs) in a dose-dependent manner. Previous PK studies demonstrated a significant increase of ectocervical and vaginal tissue MIV-150 concentrations 14 days versus 1 day post-IVR insertion, with the highest increase in vaginal tissue. Therefore, we tested PD of MIV-150 in tissues 14 days post-MIV-150 IVR insertion. Ex vivo SHIV-RT infection of vaginal, but not ectocervical, tissues collected 14 days post-MIV-150 IVR insertion was significantly inhibited compared to infection at the baseline (prior to MIV-150 IVR exposure). No changes in vaginal and ectocervical tissue infection were observed after placebo IVR exposure. Overall, these data underscore the use of the ex vivo macaque explant challenge models to evaluate tissue and VF PK/PD of candidate microbicides before in vivo animal efficacy studies. The data support further development of MIV-150-containing IVRs.
doi:10.1128/AAC.01529-13
PMCID: PMC3993268  PMID: 24614384
7.  Short Communication: A Repeated Simian Human Immunodeficiency Virus Reverse Transcriptase/Herpes Simplex Virus Type 2 Cochallenge Macaque Model for the Evaluation of Microbicides 
AIDS Research and Human Retroviruses  2014;30(11):1117-1124.
Abstract
Epidemiological studies suggest that prevalent herpes simplex virus type 2 (HSV-2) infection increases the risk of HIV acquisition, underscoring the need to develop coinfection models to evaluate promising prevention strategies. We previously established a single high-dose vaginal coinfection model of simian human immunodeficiency virus (SHIV)/HSV-2 in Depo-Provera (DP)-treated macaques. However, this model does not appropriately mimic women's exposure. Repeated limiting dose SHIV challenge models are now used routinely to test prevention strategies, yet, at present, there are no reports of a repeated limiting dose cochallenge model in which to evaluate products targeting HIV and HSV-2. Herein, we show that 20 weekly cochallenges with 2–50 TCID50 simian human immunodeficiency virus reverse transcriptase (SHIV-RT) and 107 pfu HSV-2 results in infection with both viruses (4/6 SHIV-RT, 6/6 HSV-2). The frequency and level of vaginal HSV-2 shedding were significantly greater in the repeated exposure model compared to the single high-dose model (p<0.0001). We used this new model to test the Council's on-demand microbicide gel, MZC, which is active against SHIV-RT in DP-treated macaques and HSV-2 and human papillomavirus (HPV) in mice. While MZC reduced SHIV and HSV-2 infections in our repeated limiting dose model when cochallenging 8 h after each gel application, a barrier effect of carrageenan (CG) that was not seen in DP-treated animals precluded evaluation of the significance of the antiviral activity of MZC. Both MZC and CG significantly (p<0.0001) reduced the frequency and level of vaginal HSV-2 shedding compared to no gel treatment. This validates the use of this repeated limiting dose cochallenge model for testing products targeting HIV and HSV-2.
doi:10.1089/aid.2014.0207
PMCID: PMC4208605  PMID: 25354024
8.  A MIV-150/Zinc Acetate Gel Inhibits SHIV-RT Infection in Macaque Vaginal Explants 
PLoS ONE  2014;9(9):e108109.
To extend our observations that single or repeated application of a gel containing the NNRTI MIV-150 (M) and zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) inhibits vaginal transmission of simian/human immunodeficiency virus (SHIV)-RT in macaques, we evaluated safety and anti-SHIV-RT activity of MZC and related gel formulations ex vivo in macaque mucosal explants. In addition, safety was further evaluated in human ectocervical explants. The gels did not induce mucosal toxicity. A single ex vivo exposure to diluted MZC (1∶30, 1∶100) and MC (1∶30, the only dilution tested), but not to ZC gel, up to 4 days prior to viral challenge, significantly inhibited SHIV-RT infection in macaque vaginal mucosa. MZC's activity was not affected by seminal plasma. The antiviral activity of unformulated MIV-150 was not enhanced in the presence of ZA, suggesting that the antiviral activity of MZC was mediated predominantly by MIV-150. In vivo administration of MZC and CG significantly inhibited ex vivo SHIV-RT infection (51–62% inhibition relative to baselines) of vaginal (but not cervical) mucosa collected 24 h post last gel exposure, indicating barrier effect of CG. Although the inhibitory effect of MZC (65–74%) did not significantly differ from CG (32–45%), it was within the range of protection (∼75%) against vaginal SHIV-RT challenge 24 h after gel dosing. Overall, the data suggest that evaluation of candidate microbicides in macaque explants can inform macaque efficacy and clinical studies design. The data support advancing MZC gel for clinical evaluation.
doi:10.1371/journal.pone.0108109
PMCID: PMC4178065  PMID: 25259616
9.  Assessment of facility readiness and provider preparedness for dealing with postpartum haemorrhage and pre-eclampsia/eclampsia in public and private health facilities of northern Karnataka, India: a cross-sectional study 
Background
The maternal mortality ratio in India has been declining over the past decade, but remains unacceptably high at 212 per 100,000 live births. Postpartum haemorrhage (PPH) and pre- eclampsia/eclampsia contribute to 40% of all maternal deaths. We assessed facility readiness and provider preparedness to deal with these two maternal complications in public and private health facilities of northern Karnataka state, south India.
Methods
We undertook a cross-sectional study of 131 primary health centres (PHCs) and 148 higher referral facilities (74 public and 74 private) in eight districts of the region. Facility infrastructure and providers’ knowledge related to screening and management of complications were assessed using facility checklists and test cases, respectively. We also attempted an audit of case sheets to assess provider practice in the management of complications. Chi square tests were used for comparing proportions.
Results
84.5% and 62.9% of all facilities had atleast one doctor and three nurses, respectively; only 13% of higher facilities had specialists. Magnesium sulphate, the drug of choice to control convulsions in eclampsia was available in 18% of PHCs, 48% of higher public facilities and 70% of private facilities. In response to the test case on eclampsia, 54.1% and 65.1% of providers would administer anti-hypertensives and magnesium sulphate, respectively; 24% would administer oxygen and only 18% would monitor for magnesium sulphate toxicity. For the test case on PPH, only 37.7% of the providers would assess for uterine tone, and 40% correctly defined early PPH. Specialists were better informed than the other cadres, and the differences were statistically significant. We experienced generally poor response rates for audits due to non-availability and non-maintenance of case sheets.
Conclusions
Addressing gaps in facility readiness and provider competencies for emergency obstetric care, alongside improving coverage of institutional deliveries, is critical to improve maternal outcomes. It is necessary to strengthen providers’ clinical and problem solving skills through capacity building initiatives beyond pre-service training, such as through onsite mentoring and supportive supervision programs. This should be backed by a health systems response to streamline staffing and supply chains in order to improve the quality of emergency obstetric care.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2393-14-304) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2393-14-304
PMCID: PMC4161844  PMID: 25189169
Postpartum haemorrhage; Pre-eclampsia; Eclampsia; Facility readiness; Provider preparedness; Quality; Maternal care; Public sector; Private sector
10.  An Appraisal of Female Sex Work in Nigeria - Implications for Designing and Scaling Up HIV Prevention Programmes 
PLoS ONE  2014;9(8):e103619.
Background
The HIV epidemic in Nigeria is complex with diverse factors driving the epidemic. Accordingly, Nigeria's National Agency for the Control of AIDS is coordinating a large-scale initiative to conduct HIV epidemic appraisals across all states. These appraisals will help to better characterize the drivers of the epidemic and ensure that the HIV prevention programmes match the local epidemic context, with resources allocated to interventions that have the greatest impact locally. Currently, the mapping and size estimation of Female Sex Workers (FSWs) - a major component of the appraisal has been completed in seven states. These states are using the data generated to plan, prioritize and scale-up sub-national HIV prevention programmes.
Methodology
It involved a two-level process of identifying and validating locations where FSWs solicit and/or meet clients (“hotspots”). In the first level, secondary key informants were interviewed to collect information about the geographic location and description of the hotspots. For the second level, FSWs were interviewed at each hotspot and information on population size estimates, typologies and operational dynamics of the FSWs were collected.
Results
Across the seven states, a total of 17,266 secondary key informants and 5,732 FSWs were interviewed. 10,233 hotspots were identified with an estimated 126,489 FSWs ranging from 5,920 in Anambra to 46,691 in Lagos. The most common hotspots were bars/nightclubs (30%), hotels/lodges (29.6%), streets (16.6%), and brothels (14.6%). Furthermore, the population density of FSWs (per thousand adult men) across the states ranged from 2 in Anambra to 17 in the Federal Capital Territory.
Conclusion
FSW populations in Nigeria are large and diverse, with substantial differences between and within states. Improved understanding of the location, population size, density, organizational typologies and clients of sex work has informed and is central to Nigeria's planning process for scaling up focused HIV prevention programmes.
doi:10.1371/journal.pone.0103619
PMCID: PMC4131880  PMID: 25118691
11.  Validation of the Modes of Transmission Model as a Tool to Prioritize HIV Prevention Targets: A Comparative Modelling Analysis 
PLoS ONE  2014;9(7):e101690.
Background
The static Modes of Transmission (MOT) model predicts the annual fraction of new HIV infections acquired across subgroups (MOT metric), and is used to focus HIV prevention. Using synthetic epidemics via a dynamical model, we assessed the validity of the MOT metric for identifying epidemic drivers (behaviours or subgroups that are sufficient and necessary for HIV to establish and persist), and the potential consequence of MOT-guided policies.
Methods and Findings
To generate benchmark MOT metrics for comparison, we simulated three synthetic epidemics (concentrated, mixed, and generalized) with different epidemic drivers using a dynamical model of heterosexual HIV transmission. MOT metrics from generic and complex MOT models were compared against the benchmark, and to the contribution of epidemic drivers to overall HIV transmission (cumulative population attributable fraction over t years, PAFt). The complex MOT metric was similar to the benchmark, but the generic MOT underestimated the fraction of infections in epidemic drivers. The benchmark MOT metric identified epidemic drivers early in the epidemics. Over time, the MOT metric did not identify epidemic drivers. This was not due to simplified MOT models or biased parameters but occurred because the MOT metric (irrespective of the model used to generate it) underestimates the contribution of epidemic drivers to HIV transmission over time (PAF5–30). MOT-directed policies that fail to reach epidemic drivers could undermine long-term impact on HIV incidence, and achieve a similar impact as random allocation of additional resources.
Conclusions
Irrespective of how it is obtained, the MOT metric is not a valid stand-alone tool to identify epidemic drivers, and has limited additional value in guiding the prioritization of HIV prevention targets. Policy-makers should use the MOT model judiciously, in combination with other approaches, to identify epidemic drivers.
doi:10.1371/journal.pone.0101690
PMCID: PMC4090151  PMID: 25014543
12.  Sex Hormones Selectively Impact the Endocervical Mucosal Microenvironment: Implications for HIV Transmission 
PLoS ONE  2014;9(5):e97767.
Several studies suggest that progesterone and estrogens may affect HIV transmission in different, possibly opposing ways. Nonetheless, a direct comparison of their effects on the mucosal immune system has never been done. We hypothesize that sex hormones might impact the availability of cells and immune factors important in early stages of mucosal transmission, and, in doing so influence the risk of HIV acquisition. To test this hypothesis, we employed 15 ovarectomized rhesus macaques: 5 were treated with Depot Medroxy Progesterone Acetate (DMPA), 6 with 17-β estradiol (E2) and 4 were left untreated. All animals were euthanized 5 weeks after the initiation of hormone treatment, a time post-DMPA injection associated with high susceptibility to SIV infection. We found that DMPA-treated macaques exhibited higher expression of integrin α4β7 (α4β7) on CD4+ T cells, the gut homing receptor and a marker of cells highly susceptible to HIV, in the endocervix than did the E2-treated animals. In contrast, the frequency of CCR5+ CD4+ T cells in DMPA-treated macaques was higher than in the E2-treated group in vaginal tissue, but lower in endocervix. α4β7 expression on dendritic cells (DCs) was higher in the DMPA-treated group in the endocervical tissue, but lower in vaginal tissue and on blood DCs compared with the E2-treated animals. Soluble MAdCAM-1, the α4β7 ligand, was present in the vaginal fluids of the control and E2-treated groups, but absent in the fluids from DMPA-treated animals. Both hormones modulated the expression and release of inflammatory factors and modified the distribution of sialomucins in the endocervix. In summary, we found that sex hormones profoundly impact mucosal immune factors that are directly implicated in HIV transmission. The effect is particularly significant in the endocervix. This may increase our understanding of the potential hormone-driven modulation of HIV susceptibility and potentially guide contraceptive policies in high-risk settings.
doi:10.1371/journal.pone.0097767
PMCID: PMC4022654  PMID: 24830732
13.  A Potent Combination Microbicide that Targets SHIV-RT, HSV-2 and HPV 
PLoS ONE  2014;9(4):e94547.
Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p = 0.0187) infection of mice when 106 pfu HSV-2 were applied immediately after vaginal challenge and also when 5×103 pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×106 HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use.
doi:10.1371/journal.pone.0094547
PMCID: PMC3989196  PMID: 24740100
14.  Efficient mucosal transmissibility but limited pathogenicity of R5 SHIVSF162P3N in Chinese origin rhesus macaques 
Background
Infection of rhesus macaques (RMs) of Indian origin with SIV or SHIV provided powerful tools to study HIV-1 transmission and disease, and for testing the efficacy of novel drugs, vaccines and prevention strategies. In developing alternative nonhuman primate AIDS models for the CCR5 (R5)-tropic SHIVSF162P3N, we characterized virus transmission and infection in Chinese origin RMs.
Methods
Virologic, immunologic and pathogenic evaluations of R5 SHIVSF162P3N infection in Chinese RMs challenged intrarectally (ir) or intravaginally (ivg) were performed and compared to those previously observed in Indian origin rhesus exposed to the same inoculum dose and via similar route.
Results
R5 SHIVSF162P3N transmits efficiently across mucosal surfaces in Chinese RMs. The magnitude and kinetics of early virus dissemination following intrarectal inoculation in the Chinese macaques were similar to those observed in Indian rhesus, but a trend towards increased SHIVSF162P3N vaginal infectivity and rapid virus spread was seen in the Chinese macaques compared to the Indian origin animals. Once infected, however, set-point viremia in the ir- and ivg-infected Chinese rhesus was significantly lower and the animals survived longer compared with infected Indian rhesus.
Conclusions
The R5 SHIVSF162P3N/Chinese rhesus macaque infection model is suitable for studies of mucosal HIV-1 transmission and protection, but the high frequency of spontaneous control of chronic viremia and reduced virulence with SHIVSF162P3N in this macaque subspecies may limit its utility in studying HIV-1 pathogenesis and in evaluating vaccines and antiretrovirals that rely on reduction in chronic viral load or AIDS development as an experimental endpoint.
doi:10.1097/QAI.0b013e31827f1c11
PMCID: PMC3622143  PMID: 23221980
SHIV; rhesus macaque subspecies; mucosal transmission; AIDS
15.  Granuloma Correlates of Protection Against Tuberculosis and Mechanisms of Immune Modulation by Mycobacterium tuberculosis 
The Journal of Infectious Diseases  2012;207(7):1115-1127.
Background. The BCG vaccine is ineffective against adult tuberculosis. Hence, new antituberculosis vaccines are needed. Correlates of protection against tuberculosis are not known. We studied the effects of BCG vaccination on gene expression in tuberculosis granulomas using macaques.
Methods. Macaques were BCG-vaccinated or sham-vaccinated and then challenged with virulent Mycobacterium tuberculosis. Lung lesions were used for comparative transcriptomics.
Results. Vaccinated macaques were protected with lower bacterial burden and immunopathology. Lesions from BCG-vaccinated nonhuman primates (NHPs) showed a better balance of α- and β-chemokine gene expression with higher levels of β-chemokine expression relative to nonvaccinated animals. Consistent with this, sham-vaccinated macaques recruited fewer macrophages relative to neutrophils in their lungs. The expression of indoleamine 2,3-dioxygenase (IDO), a known immunosuppressor, was significantly higher in both week 5 and 10 lesions from sham-vaccinated, relative to BCG-vaccinated, NHPs. IDO expression was primarily limited to the nonlymphocytic region of the lesions, within the inner ring structure surrounding the central necrosis.
Conclusions. Our study defines lung gene expression correlates of protective response against tuberculosis, relative to disease, which can potentially be employed to assess the efficacy of candidate antituberculosis vaccines. Mycobacterium tuberculosis may modulate protective immune responses using diverse mechanisms, including increased recruitment of inflammatory neutrophils and the concomitant use of IDO to modulate inflammation.
doi:10.1093/infdis/jis778
PMCID: PMC3633457  PMID: 23255564
Mycobacterium tuberculosis; transcriptomics; IDO
16.  Estimating the Size of the Female Sex Worker Population in Kenya to Inform HIV Prevention Programming 
PLoS ONE  2014;9(3):e89180.
Background
The high burden of HIV infections among female sex workers (FSW) in sub-Saharan Africa has been long recognised, but effective preventive interventions have largely not been taken to scale. We undertook a national geographical mapping exercise in 2011/2012 to assess the locations and population size of FSW in Kenya, to facilitate targeted HIV prevention services for this population.
Methods and Findings
We used a geographical mapping approach, consisting of interviews with secondary key informants to identify “hot” spots frequented by FSW, their operational dynamics and the estimated numbers of FSW in those spots. This was followed by validation of the estimates through interviews with FSW at each spot identified. The mapping covered Nairobi, the capital city of Kenya, and 50 other major urban centres. In total, 11,609 secondary key informant interviews were conducted to identify FSW spots. Further, a total of 6,360 FSW were interviewed for spot validation purposes. A total of 10,670 spots where FSW congregate were identified. The estimated FSW population in all the towns mapped was 103,298 (range 77,878 to 128, 717). Size estimates in the towns mapped were extended to smaller towns that were not mapped, using a statistical model. The national urban FSW population estimate was 138,420 (range 107, 552 to 169, 288), covering all towns of over 5,000 population. We estimated that approximately 5% of the urban female population of reproductive age in Kenya could be sex workers, which is consistent with previous estimates from other sub-Saharan African countries.
Conclusions
This study provides the first national level data on the size of the FSW population in Kenya. These data can be used to enhance HIV prevention programme planning and implementation for FSW, to form the basis for impact evaluations, and to improve programme coverage by directing efforts to locations with the greatest need.
doi:10.1371/journal.pone.0089180
PMCID: PMC3940432  PMID: 24595029
17.  Exposure to MIV-150 from a High-Dose Intravaginal Ring Results in Limited Emergence of Drug Resistance Mutations in SHIV-RT Infected Rhesus Macaques 
PLoS ONE  2014;9(2):e89300.
When microbicides used for HIV prevention contain antiretroviral drugs, there is concern for the potential emergence of drug-resistant HIV following use in infected individuals who are either unaware of their HIV infection status or who are aware but still choose to use the microbicide. Resistant virus could ultimately impact their responsiveness to treatment and/or result in subsequent transmission of drug-resistant virus. We tested whether drug resistance mutations (DRMs) would emerge in macaques infected with simian immunodeficiency virus expressing HIV reverse transcriptase (SHIV-RT) after sustained exposure to the potent non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 delivered via an intravaginal ring (IVR). We first treated 4 SHIV-RT-infected animals with daily intramuscular injections of MIV-150 over two 21 day (d) intervals separated by a 7 d drug hiatus. In all 4 animals, NNRTI DRMs (single and combinations) were detected within 14 d and expanded in proportion and diversity with time. Knowing that we could detect in vivo emergence of NNRTI DRMs in response to MIV-150, we then tested whether a high-dose MIV-150 IVR (loaded with >10 times the amount being used in a combination microbicide IVR in development) would select for resistance in 6 infected animals, modeling use of this prevention method by an HIV-infected woman. We previously demonstrated that this MIV-150 IVR provides significant protection against vaginal SHIV-RT challenge. Wearing the MIV-150 IVR for 56 d led to only 2 single DRMs in 2 of 6 animals (430 RT sequences analyzed total, 0.46%) from plasma and lymph nodes despite MIV-150 persisting in the plasma, vaginal fluids, and genital tissues. Only wild type virus sequences were detected in the genital tissues. These findings indicate a low probability for the emergence of DRMs after topical MIV-150 exposure and support the advancement of MIV-150-containing microbicides.
doi:10.1371/journal.pone.0089300
PMCID: PMC3937329  PMID: 24586674
18.  Modelling the interactions between Herpes simplex virus type-2 (HSV-2) and HIV: implications for the HIV epidemic in Southern India 
Background
The role of HSV-2 in the HIV epidemic, and the potential impact of HSV-2 suppressive therapy, have been previously explored only within the context of sub-Saharan Africa. In this analysis, modelling is used to estimate: (1) the contribution of herpes simplex virus type-2 (HSV-2) to HIV transmission from clients to female sex workers (FSWs) in a Southern Indian setting; and (2) the maximum potential impact of ‘perfect’ HSV-2 suppressive therapy on HIV incidence.
Methods
A dynamic HSV-2/HIV model was developed, parameterised and fitted to Mysore data. The model estimated the attributable fractions (AFs) of HIV infections due to HSV-2. Multivariate sensitivity analyses and regression analyses were conducted.
Results
The model suggests that 36% (95% CI: 22-62%) of FSW HIV infections were due to HSV-2, mostly through HSV-2 asymptomatic shedding. Even if HSV-2 suppressive therapy could eliminate the effect of HSV-2 on HIV infectivity among all coinfected clients, only 15% (95% CI: 3-41%) of HIV infections among FSWs would have been averted. 36% (95% CI: 18-61%) of HIV infections among HSV-2-infected FSWs could have been averted if suppressive therapy reduced their risk of HIV acquisition to that of the HSV-2-uninfected FSWs.
Conclusions
HSV-2 contributes substantially to HIV in this Southern Indian context. However, even in the best case scenario, HSV-2 suppressive therapy is unlikely to reduce HIV transmission or acquisition by more than 50% (as aimed for in recent trials), because of the limited strength of the interaction effect between HSV-2 and HIV.
doi:10.1136/sti.2009.041699
PMCID: PMC3920054  PMID: 21059838
HIV; Herpes simplex virus type-2; mathematical modelling; India; female sex work
19.  A Combination Microbicide Gel Protects Macaques Against Vaginal Simian Human Immunodeficiency Virus-Reverse Transcriptase Infection, But Only Partially Reduces Herpes Simplex Virus-2 Infection After a Single High-Dose Cochallenge 
Abstract
Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.
doi:10.1089/aid.2013.0165
PMCID: PMC3910668  PMID: 24117013
20.  The potential of liposome–encapsulated ciprofloxacin as a tularemia therapy 
Liposome-encapsulation has been suggested as method to improve the efficacy of ciprofloxacin against the intracellular pathogen, Francisella tularensis. Early work with a prototype formulation, evaluated for use against the F. tularensis live vaccine strain, showed that a single dose of liposomal ciprofloxacin given by the intranasal or inhalational route could provide protection in a mouse model of pneumonic tularemia. Liposomal ciprofloxacin offered better protection than ciprofloxacin given by the same routes. Liposomal ciprofloxacin has been further developed by Aradigm Corporation for Pseudomonas aeruginosa infections in patients with cystic fibrosis and non-cystic fibrosis bronchiectasis. This advanced development formulation is safe, effective and well tolerated in human clinical trials. Further evaluation of the advanced liposomal ciprofloxacin formulation against the highly virulent F. tularensis Schu S4 strain has shown that aerosolized CFI (Ciprofloxacin encapsulated in liposomes for inhalation) provides significantly better protection than oral ciprofloxacin. Thus, liposomal ciprofloxacin is a promising treatment for tularemia and further research with the aim of enabling licensure under the animal rule is warranted.
doi:10.3389/fcimb.2014.00079
PMCID: PMC4062069  PMID: 24995163
tularemia; liposomal; ciprofloxacin; Francisella tularensis
21.  Generation of Lineage-Related, Mucosally Transmissible Subtype C R5 Simian-Human Immunodeficiency Viruses Capable of AIDS Development, Induction of Neurological Disease, and Coreceptor Switching in Rhesus Macaques 
Journal of Virology  2013;87(11):6137-6149.
Most human immunodeficiency virus (HIV) transmissions are initiated with CCR5 (R5)-using viruses across mucosal surfaces, with the majority in regions where HIV type 1 (HIV-1) clade C predominates. Mucosally transmissible, highly replication competent, pathogenic R5 simian-human immunodeficiency viruses (SHIVs) encoding biologically relevant clade C envelopes are therefore needed as challenge viruses in vaccine efficacy studies with nonhuman primates. Here we describe the generation of three lineage-related subtype C SHIVs through four successive rapid transfers in rhesus macaques of SHIVC109F.PB4, a molecular clone expressing the soluble-CD4 (sCD4)-sensitive CCR5-tropic clade C envelope of a recently infected subject in Zambia. The viruses differed in their monkey passage histories and neutralization sensitivities but remained R5 tropic. SHIVC109P3 and SHIVC109P3N were recovered from a passage-3 rapid-progressor animal during chronic infection (24 weeks postinfection [wpi]) and at end-stage disease (34 wpi), respectively, and are classified as tier 1B strains, whereas SHIVC109P4 was recovered from a passage-4 normal-progressor macaque at 22 wpi and is a tier 2 virus, more difficult to neutralize. All three viruses were transmitted efficiently via intrarectal inoculation, reaching peak viral loads of 107 to 109 RNA copies/ml plasma and establishing viremia at various set points. Notably, one of seven (GC98) and two of six (CL31, FI08) SHIVC109P3- and SHIVC109P3N-infected macaques, respectively, progressed to AIDS, with neuropathologies observed in GC98 and FI08, as well as coreceptor switching in the latter. These findings support the use of these new SHIVC109F.PB4-derived viruses to study the immunopathology of HIV-1 clade C infection and to evaluate envelope-based AIDS vaccines in nonhuman primates.
doi:10.1128/JVI.00178-13
PMCID: PMC3648099  PMID: 23514895
22.  Understanding out-migration among female sex workers in South India 
Sexually transmitted diseases  2012;39(10):776-783.
BACKGROUND
Migrant sex workers are known to be vulnerable to HIV. There is substantial female sex worker (FSW) mobility between the borders of Maharashtra and Karnataka, but little programming emphasis on migrant FSWs in India. We sought to understand the individual/cultural, structural and contextual determinants of migration among FSWs from Karnataka.
METHODS
A cross sectional face-to-face interview of 1567FSWs from 142 villages in 3 districts of northern Karnataka, India was conducted from January–June 2008. Villages having 10+FSWs, a large number of whom were migrant, were selected following mapping of FSWs. Multinomial logistic regression was conducted to identify characteristics associated with migrant (travelled for ≥2weeks outside the district past year) and mobile (travelled for <2weeks outside the district past year) FSWs; adjusting for age and district.
RESULTS
Compared to non-migrants, migrant FSWs were more likely to be brothel than street-based (AOR 5.7; 95%CI 1.6–20.0), have higher income from sex work (AOR 42.2; 12.6–142.1), speak >2languages (AOR 5.6%; 2.6–12.0), have more clients (AORper client 2.9; 1.2–7.2) and have more sex acts/day (AORper sex act 3.5; 1.3–9.3). Mobile FSWs had higher income from sex work (AOR=13.2; 3.9–44.6) relative to non-migrants, but not as strongly as for migrant FSWs.
CONCLUSION
Out-migration of FSWs in Karnataka was strongly tied to sex work characteristics; thus, the structure inherent in sex work should be capitalized on when developing HIV preventive interventions. The important role of FSWs in HIV epidemics, coupled with the potential for rapid spread of HIV with migration, requires the most effective interventions possible for mobile and migrant FSWs.
doi:10.1097/OLQ.0b013e3182641d90
PMCID: PMC3457025  PMID: 23001264
Migration; HIV; India; Female sex worker
23.  Myocarditis, Disseminated Infection, and Early Viral Persistence Following Experimental Coxsackievirus B Infection of Cynomolgus Monkeys 
PLoS ONE  2013;8(9):e74569.
Coxsackievirus B (CVB) infection is a common cause of acute viral myocarditis. The clinical presentation of myocarditis caused by this enterovirus is highly variable, ranging from mildly symptoms to complete hemodynamic collapse. These variations in initial symptoms and in the immediate and long term outcomes of this disease have impeded development of effective treatment strategies. Nine cynomolgus monkeys were inoculated with myocarditic strains of CVB. Virological studies performed up to 28 days post-inoculation demonstrated the development of neutralizing antibody in all animals, and the presence of CVB in plasma. High dose intravenous inoculation (n = 2) resulted in severe disseminated disease, while low dose intravenous (n = 6) or oral infection (1 animal) resulted in clinically unapparent infection. Transient, minor, echocardiographic abnormalities were noted in several animals, but no animals displayed signs of significant acute cardiac failure. Although viremia rapidly resolved, signs of myocardial inflammation and injury were observed in all animals at the time of necropsy, and CVB was detected in postmortem myocardial specimens up to 28 days PI. This non-human primate system replicates many features of illness in acute coxsackievirus myocarditis and demonstrates that myocardial involvement may be common in enteroviral infection; it may provide a model system for testing of treatment strategies for enteroviral infections and acute coxsackievirus myocarditis.
doi:10.1371/journal.pone.0074569
PMCID: PMC3767629  PMID: 24040287
24.  A Modified Zinc Acetate Gel, a Potential Nonantiretroviral Microbicide, Is Safe and Effective against Simian-Human Immunodeficiency Virus and Herpes Simplex Virus 2 Infection In Vivo 
We previously showed that a prototype gel comprising zinc acetate (ZA) in carrageenan (CG) protected mice against vaginal and rectal herpes simplex virus 2 (HSV-2) challenge as well as macaques against vaginal simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) challenge. In this work, we modified buffers and cosolvents to obtain a stable, nearly iso-osmolal formulation and evaluated its safety and efficacy against SHIV-RT and HSV-2. In vitro toxicity to lactobacilli and Candida albicans was determined. Macaques were given daily doses of ZA and CG (ZA/CG) or CG alone vaginally for 14 days and challenged with SHIV-RT 24 h later. Mice were challenged vaginally or rectally with HSV-2 immediately after a single gel treatment to measure efficacy or vaginally 12 h after daily gel treatment for 7 days to evaluate the gel's impact on susceptibility to HSV-2 infection. The modified ZA/CG neither affected the viability of lactobacilli or C. albicans nor enhanced vaginal HSV-2 infection after daily ZA/CG treatment. Vaginal SHIV-RT infection of macaques was reduced by 66% (P = 0.006) when macaques were challenged 24 h after the last dose of gel. We observed 60% to 80% uninfected mice after vaginal (P < 0.0001) and rectal (P = 0.008) high-dose HSV-2 challenge. The modified ZA/CG gel is safe and effective in animal models and represents a potential candidate to limit the transmission of HIV and HSV-2.
doi:10.1128/AAC.00796-13
PMCID: PMC3719770  PMID: 23752515
25.  Serological survey of the novel influenza A H1N1 in inner city Winnipeg, Manitoba, 2009 
INTRODUCTION:
Little is known about the determinants of pandemic H1N1 (pH1N1) infection in Canada among low-income, inner city populations. To inform future influenza planning, the seroprevalence of pH1N1 antibodies among inner city clinic attendees in Winnipeg (Manitoba) according to sociodemographic and risk factor characteristics were estimated and vaccination rates were explored.
METHODS:
Adults presenting to three inner city community clinics in Winnipeg from October 2009 to December 2009 were recruited as study participants (n=458). A questionnaire was administered to collect demographic, risk factor and symptom information, and a venous blood sample was collected for hemagglutination inhibition assay testing to detect the presence of antibodies against pH1N1.
RESULTS:
Approximately one-half (53%) of the study participants reported an annual household income of <$10,000/year, and 65% identified as Aboriginal. pH1N1 positivity was 5.7% among those enrolled early in the study and 15.5% among those enrolled later in the study. Positivity was higher among participants who were female, Aboriginal and in contact with children ≤5 years of age. The overall pH1N1 vaccination rate was 28%.
DISCUSSION:
pH1N1 positivity was high among low-income adults accessing clinics in Winnipeg’s inner city compared with the general population. Of further concern were the low rates of uptake of both seasonal and pH1N1 influenza vaccinations. When planning for future influenza outbreaks, it is important to incorporate strategies for the prevention, control, and care of influenza among low-income and inner city adults.
PMCID: PMC3403663  PMID: 23730311
Epidemiology; Hemagglutination inhibition assay; Inner city; Pandemic Influenza A H1N1; pH1N1; Serological survey

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