Sex worker-led structural interventions in India: a case study on addressing violence in HIV prevention through the Ashodaya Samithi collective in Mysore.

BACKGROUND & OBJECTIVES: Structural interventions have the capacity to improve the outcomes of HIV/AIDS interventions by changing the social, economic, political or environmental factors that determine risk and vulnerability. Marginalized groups face disproportionate barriers to health, and sex workers are among those at highest risk of HIV in India. Evidence in India and globally has shown that sex workers face violence in many forms ranging from verbal, psychological and emotional abuse to economic extortion, physical and sexual violence and this is directly linked to lower levels of condom use and higher levels of sexually transmitted infections (STIs), the most critical determinants of HIV risk. We present here a case study of an intervention that mobilized sex workers to lead an HIV prevention response that addresses violence in their daily lives. METHODS: This study draws on ethnographic research and project monitoring data from a community-led structural intervention in Mysore, India, implemented by Ashodaya Samithi. Qualitative and quantitative data were used to characterize baseline conditions, community responses and subsequent outcomes related to violence. RESULTS: In 2004, the incidence of reported violence by sex workers was extremely high (> 8 incidents per sex worker, per year) but decreased by 84 per cent over 5 years. Violence by police and anti-social elements, initially most common, decreased substantially after a safe space was established for sex workers to meet and crisis management and advocacy were initiated with different stakeholders. Violence by clients, decreased after working with lodge owners to improve safety. However, initial increases in intimate partner violence were reported, and may be explained by two factors: (i) increased willingness to report such incidents; and (ii) increased violence as a reaction to sex workers' growing empowerment. Trafficking was addressed through the establishment of a self-regulatory board (SRB). The community's progressive response to violence was enabled by advancing community mobilization, ensuring community ownership of the intervention, and shifting structural vulnerabilities, whereby sex workers increasingly engaged key actors in support of a more enabling environment. INTERPRETATION & CONCLUSIONS: Ashodaya's community-led response to violence at multiple levels proved highly synergistic and effective in reducing structural violence.

Characterization of antibody responses to combinations of a dengue-2 DNA and dengue-2 recombinant subunit vaccine.

A dengue-2 (DEN-2) DNA vaccine coding for the premembrane and envelope (E) proteins and a recombinant fusion protein containing the B domain of the DEN-2 E protein fused to the maltose-binding protein (MBP) of Escherichia coli both elicited neutralizing antibody in mice. In order to achieve more rapid protective immunity as well as to increase the persistence of neutralizing antibody, we primed mice with the DNA vaccine (D), the recombinant MBP protein (R), or both (RD) given simultaneously, and then boosted twice with either the R (R/R/R or D/R/R) or D (D/D/D or R/D/D) constructs alone or the RD (RD/RD/RD) combination. All of the recombinant protein vaccines were given with alum as an adjuvant. The serum antibody response measured by enzyme-linked immunosorbent assay was highest in D/D/D mice and RD/RD/RD mice. The D/R/R mice showed an intermediate response, and the R/D/D and R/R/R showed the lowest response. The geometric mean (GM) 50% neutralizationtiter (50% plaque reduction neutralization, or PRNT50) was marginally higher for RD/RD/RD mice (891) at 9 months after priming than that for R/R/R mice (724). T he lowest GM PRNT50 titers were seen in the D/D/D mice (33) and R/D/D mice (40), and the D/R/R group had a slightly higher titer (156) than these 2 groups. The predominant antibody subclass for the D/D/D mice was immunoglobulin (Ig) G2a, similar to mice infected with live virus. The R/R/R mice showed an exclusive IgGI antibody response, and the RD/RD/RD response also was predominantly IgGI. The antibody subclass pattern of the R/D/D and D/R/R mice showed a more balanced distribution of both IgG1 and IgG2a. Investigating the neutralizing capacity of antibody subclasses suggested that both IgG1 and IgG2a could neutralize DEN-2 virus. Our observations indicate that the combination RD prime-boost regimen warrants further investigation as a vaccine strategy to prevent dengue infection.

Synergistic neutralizing antibody response to a dengue virus type 2 DNA vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing GM-CSF.

We have previously shown that a dengue virus type 1 DNA vaccine expressing premembrane (prM) and envelope (E) genes was immunogenic in mice and monkeys and that rhesus monkeys vaccinated with this construct were completely to partially protected from virus challenge. In order to improve the immunogenicity of dengue DNA vaccines, we have evaluated the effect of lysosome targeting of antigens and coimmunization with a plasmid expressing GM-CSF on antibody responses. A dengue virus type 2 candidate vaccine containing prM and E genes was constructed in which the transmembrane and cytoplasmic regions of E were replaced by those of the lysosome-associated membrane protein (LAMP). The modified vaccine construct expressed antigen that was colocalized with endogenous LAMP in lysosomal vesicles of transfected cells, whereas the antigen expressed from the unmodified construct was not. It was hypothesized that targeting of antigen to the lysosomal compartment will increase antigen presentation by MHC class II, leading to stronger CD4-mediated immune responses. Mice immunized with the modified construct responded with significantly higher levels of virus neutralizing antibodies compared to those immunized with the unmodified construct. Coimmunization of mice with a plasmid expressing murine GM-CSF enhanced the antibody response obtained with either the unmodified or the modified construct alone. The highest antibody responses were noted when the modified construct was coinjected with plasmid expressing the GM-CSF gene. These results could form the basis for an effective tetravalent dengue virus DNA vaccine.

Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques.

A candidate DNA vaccine expressing dengue virus type 1 pre-membrane and envelope proteins was used to immunize rhesus macaques. Monkeys were immunized intramuscularly (i.m.) or intradermally (i.d.) by three or four 1 mg doses of vaccine, respectively. Monkeys that were inoculated i.m. seroconverted more quickly and had higher antibody levels than those that were inoculated i.d. The sera exhibited virus-neutralizing activity, which declined over time. Four of the eight i.m.-inoculated monkeys were protected completely from developing viraemia when challenged 4 months after the last dose with homologous dengue virus. The other four monkeys had reduced viraemia compared with the control immunized monkeys. The i.d. -inoculated monkeys showed no reduction in viraemia when challenged with the virus. All vaccinated monkeys showed an anamnestic antibody response, indicating that they had established immunological memory. Vaccine-induced antibody had an avidity index similar to that of antibody induced by virus infection; however, no clear correlation was apparent between antibody avidity and virus neutralization titres.

A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys.

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the naïve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.

Immunogenicity of dengue virus type 1 DNA vaccines expressing truncated and full length envelope protein.

Recombinant plasmid DNA constructs expressing truncated or full-length dengue-1 envelope (E) with or without the pre-membrane (prM) were tested for immunogenicity in mice, as candidate dengue DNA vaccines. Two plasmids, one expressing the N-terminal 80% E and the other expressing prM and full length E were immunogenic in intradermally inoculated mice. The vaccinated mice produced dengue-1 specific antibodies that were both neutralizing and long lasting. Data suggested that the plasmid expressing prM and full length E produced virus like particles in transfected cells, and is probably a better immunogen compared to that expressing 80% E.

Protective efficacy of a dengue 2 DNA vaccine in mice and the effect of CpG immuno-stimulatory motifs on antibody responses.

A recently described DNA vaccine for dengue (DEN) type 2 was shown to elicit high levels of neutralizing antibodies in mice. The vaccine candidate consists of the PreM and 92% of the envelope genes of DEN 2 New Guinea C strain. We further evaluated this DNA vaccine candidate by examining the effect of immuno-stimulatory CpG DNA motifs on antibody response and by studying the protective efficacy of the vaccine. The results showed that CpG motifs present in pUC 19 significantly improved the antibody response to a suboptimal dose of 3.1 micrograms of the DEN DNA vaccine. In a lethal mouse intracerebral challenge model, the vaccine provided a significant level of protection. Sixty percent of the mice immunized with the DEN DNA vaccine plus pUC 19 survived the challenge compared to only 10% in the control group that received vector plus pUC. These studies illustrate that nucleic acid immunization is a viable approach to developing a DEN vaccine and that immuno-stimulatory CpG DNA motifs can be used to lower the minimum dose required to produce an antibody response.

Conversion of dengue virus replicative form RNA (RF) to replicative intermediate (RI) by nonstructural proteins NS-5 and NS-3.

Dengue viruses infect more than 100 million people each year and cause serious clinical manifestations. It is important to understand the replication of these viruses so that therapeutic and/or prophylactic agents may be designed. Dengue virus type 2 nonstructural proteins NS-5 and NS-3 were produced by in vitro transcription and translation of cloned genes. Both proteins possessed RNA-dependent RNA polymerase activity as measured by their ability to convert purified replicative form (RF) RNA to replicative intermediate (RI). The recombinant proteins, however, required one or more cellular protein(s) for their activity. Examination of NS-3 protein sequence revealed heretofore unnoticed sequence similarities with other polymerases.

Inoculation of plasmids expressing the dengue-2 envelope gene elicit neutralizing antibodies in mice.

To develop a nucleic acid vaccine against dengue type-2 virus, the PreM and 92% of the envelope (E) genes were cloned into different eukaryotic plasmid expression vectors (pkCMVint Polyli and pVR1012). The resultant plasmid constructs (pD2ME and P1012D2ME) properly expressed the truncated E protein in vitro as evidenced by the expected protein size on SDS-PAGE and the ability of the protein to be recognized by monoclonal antibodies directed against conformational epitopes. Three-week-old BALB/c mice were given intradermal inoculations of each construct. Plasmid expression vectors without dengue genes were used as controls. One hundred percent of the mice that received the pD2ME and p1012D2ME constructs developed anti-dengue antibodies. These antibodies were shown to neutralize dengue type-2 virus in vitro. This is the first demonstration of the use of nucleic acid inoculation in the development of potential dengue virus. vaccines.

Inhibition of dengue virus by novel, modified antisense oligonucleotides.

Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex.

Effects of deletions in the carboxy-terminal hydrophobic region of herpes simplex virus glycoprotein gB on intracellular transport and membrane anchoring.

The gB glycoprotein of herpes simplex virus type 1 is involved in viral entry and fusion and contains a predicted membrane-anchoring sequence of 69 hydrophobic amino acids, which can span the membrane three times, near the carboxy terminus. To define the membrane-anchoring sequence and the role of this hydrophobic stretch, we have constructed deletion mutants of gB-1, lacking one, two, or three predicted membrane-spanning segments within the 69 amino acids. Expression of the wild-type and mutant glycoproteins in COS-1 cells show that mutant glycoproteins lacking segment 3 (amino acids 774 to 795 of the gB-1 protein) were secreted from the cells. Protease digestion and alkaline extraction of microsomes containing labeled mutant proteins further showed that segment 3 was sufficient for stable membrane anchoring of the glycoproteins, indicating that this segment may specify the transmembrane domain of the gB glycoprotein. Also, the mutant glycoproteins containing segment 3 were localized in the nuclear envelop, which is the site of virus budding. Deletion of any of the hydrophobic segments, however, affected the intracellular transport and processing of the mutant glycoproteins. The mutant glycoproteins, although localized in the nuclear envelope, failed to complement the gB-null virus (K082). These results suggest that the carboxy-terminal hydrophobic region contains essential structural determinants of the functional gB glycoprotein.

Acid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD.

Enveloped animal viruses enter host cells either by direct fusion at neutral pH or by endocytosis. Herpes simplex virus (HSV) is believed to fuse with the plasma membrane of cells at neutral pH, and the glycoproteins gB and gD have been implicated in virus entry and cell fusion. Using cloned gB or gD genes, we show that cells expressing HSV-1 glycoproteins gB or gD can undergo fusion to form polykaryons by exposure only to acidic pH. The low pH-induced cell fusion was blocked in the presence of monoclonal antibodies specific to the glycoproteins. Infection of cells expressing gB or gD glycoproteins with HSV-1 inhibited the low pH-induced cell fusion. The results suggest that although the glycoproteins gB and gD possess fusogenic activity at acidic pH, other HSV proteins may regulate it such that in the virus-infected cell, this fusion activity is blocked.

Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein.

Herpes simplex virus (HSV) buds from the inner nuclear membrane of the infected cells. The glycoprotein gB-1 of HSV contains a stretch of 69 hydrophobic amino acids near the COOH terminus and a 109-amino acid cytoplasmic domain. By oligonucleotide-directed mutagenesis, five gB-1 mutants were constructed which either lack a cytoplasmic tail or contained 3, 6, 22, or 43 amino acids in the cytoplasmic tail. When expressed in COS cells all of the mutant glycoproteins were synthesized but the rate of intracellular transport and the appearance at the cell surface of the mutant gB-1 protein lacking the cytoplasmic tail or containing 3 and 6 amino acids in the cytoplasmic domain was drastically reduced. The wild-type gB-1 as well as all of the mutants in the cytoplasmic tail were, however, located on the nuclear envelope. These results suggest that the cytoplasmic domain of the glycoprotein gB may play a role in intracellular transport but not in the nuclear localization.

The mouse adenovirus type 1 contains an unusual E3 region.

Since the E3 region of human adenoviruses codes for a series of proteins that are probably involved in viral pathogenesis, the nucleotide sequence for a 3.6-kilobase DNA fragment in the corresponding region (map units 77 through 89) of the mouse adenovirus type 1 genome has been determined. Analysis of the sequence revealed that the genes for the fiber and for the precursor to the hexon-associated protein, pVIII, that usually flank the E3 region, are well conserved. However, many of the open reading frames contained in the E3 region of human adenoviruses between the pVIII and the fiber genes were absent from the mouse adenovirus type 1 genome.

2'-O-methyl-1-methyl adenosine: a new modified nucleoside in ragi (Eleusine coracana) tRNA.

A new modified nucleoside 2'-O-methyl-l-methyl adenosine has been found to be present in the tRNA of Eleusine coracana ( ragi ) seedlings. The sequence of the dinucleotide of which this modified nucleoside is a part suggests its presence in phenylalanine-tRNA. The structural implications of the presence of this new modification are discussed.