Due to the establishment of the National Access to Antiretroviral Program for People who have AIDS (NAPHA), approximately 80,000 Thai HIV-1 infected patients received antiretroviral drugs through the NAPHA program, which was completed at the end of 2005. The development of drug resistance is required for access to ARV drugs. The objective of this study was to determine the prevalence of antiretroviral drug resistance in Thai HIV-1 treated individuals after completing the NAPHA program.

To determine the prevalence of antiretroviral resistance in treatment-failure HIV- 1 infected individuals, under the initiative of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimen in Thailand, plasma samples were collected from 1,376 HIV-1 infected patients, who were failing in their current HAART therapy during 2000-2004. They were stratified into 2 intervals: group one (1), 558 HIV-1 infected patients (2000-2002; before the initiative of access to HAART), and group two (2), 818 HIV-1 infected patients (2003-2004; after the initiative of access to HAART). Genotypic resistance testing was performed. The frequency of antiretroviral drug resistance in treatment-failure HIV-1 infected patients has significantly increased over time from 68.5% (382/558) during 2000-2002 to 74.9% (613/818) during 2003-2004 (P<0.01). Resistance to NNRTI during 2003-2004 (59.2%) was much higher than that during 2000-2002 (36.9%; P<0.001). However, the frequency of nucleoside reverse transcriptase inhibitor (NRTI) drug resistance was not significantly higher (P=0.153). We showed that this correlated with an increase in the NNRTI-based regimen prescribed during 2003-2004, especially the Thai-produced combination pill, GPO-VIR. Our finding also showed that a high level of genotypic drug resistance is associated with GPO-VIR (40.8% lamivudine, 40.6% stavudine, 43.8% nevirapine). In order to avoid the rapid emergence of resistant viruses in a resource-poor setting, a close surveillance of antiretroviral drug resistance is feasible and should be considered.

ObjectivesTo observe the long‐term effects of an immune‐based therapy HIV‐1 Immunogen (REMUNE®; Immune Response Corp., Carlsbad, CA, USA) as a first course of treatment designed to sustain the immune system and thus delay the initiation of therapy with antiretroviral drugs and/or delay disease progression.MethodsIn this open‐label, multi‐institute extended phase II P2101B study, disease progression, CD4 and CD8 T‐cell counts, HIV‐1 RNA levels, and genotypic antiretroviral drug resistance were examined in 223 asymptomatic HIV‐1‐infected Thai volunteers receiving REMUNE® every 12 weeks over 132 weeks. A subset of subjects was randomly selected by the physicians to receive antiretroviral drugs for 10 months.ResultsPatients treated with REMUNE® demonstrated a low rate of clinical disease progression (0.72 per 100 person‐years), higher CD4 and CD8 T‐cell counts, higher body weight before and after treatment in the same patient, and stable viral load with no serious adverse events. We found no genotypic evidence of drug resistance in subgroups of patients on REMUNE® monotherapy or REMUNE® plus antiretrovirals (ARTs).ConclusionsThis Thai study, like previous US and European studies, confirms that therapeutic immunization of HIV‐infected volunteers modifies disease progression, as evidenced by stabilization of CD4 and CD8 T‐cell counts, body weight, and viral load. As the majority of asymptomatic patients demonstrated an objective response to immunization, this study suggests that REMUNE® may be utilized prior to initiation of antiviral drug therapy when CD4 cell counts are still above the current ART guidelines. Further work should be carried out to examine its potential use in combination with ART in order to reduce the increasingly common occurrence of drug resistance.

PURPOSE:The purpose of this 2-year follow-up study was to investigate the long-term effect of Remune as monotherapy for HIV-1 infection.BACKGROUND:Participants previously enrolled in the phase II double-blind, randomized, adjuvant-controlled study of the HIV-1 Immunogen (Remune) were followed for 2 years. Open-label immunization with Remune monotherapy was given to each participant every 12 weeks. Remune, a gp 120-depleted HIV-1 that was inactivated in beta-propiolactone and irradiation, was emulsified with mineral oil (incomplete Freund's adjuvant).METHOD:In Study 2101B, the effect of four doses of Remune given every 12 weeks over 40 weeks was compared to placebo in 297 asymptomatic type E HIV-infected patients [Churdboonchart et al., 2000]. A group of 17 volunteers were separated into a subset study and another 57 were excluded from analysis due to discontinuation or addition of other treatments. This 2-year follow-up study continued with open-label dosing of HIV-1 Immunogen every 12 weeks for the remaining 223 patients. Changes in CD4+ cells, CD8+ cells, and body weight were monitored at each patient visit.RESULTS:Overall, immunizations were safe; common adverse events were tolerable injection site reactions. CD4+ T-cell counts remained stable over the 132-week observation period for this cohort with a slight increase of 36.01 cells/microL. CD8+ T-cell counts showed an increase from baseline during the follow-up period (415.21 cells/microL). Furthermore, we also observed an increase in body weight from baseline (1.08 kg) at week 132. In addition, baseline CD4 count appeared to predict CD4 count at week 132 (slope = 0.31, p <.0001).CONCLUSION:These results suggest that long-term treatment of HIV-1 infection with Remune monotherapy is safe and results in a stabilization of CD4+ counts. Furthermore, it is likely that HIV-1 therapeutic immunization may show its greatest clinical benefit in participants with higher CD4+ cell counts. Such an approach may have important ramifications in developing countries where access to antiviral drugs is limited and also in early chronic HIV-1 infection when CD4+ cells are still over 300 cells/microL in order to limit the cost and toxicity.

ABSTRACT We examined the effect of a human immunodeficiency virus (HIV)-specific immune-based therapy in Thailand, where access to antiviral drug therapy is limited. A 40-week trial was conducted with 297 asymptomatic, HIV-infected Thai subjects with CD4-cell counts greater than 300 μl/mm 3 . Subjects were randomized to receive either HIV type 1 (HIV-1) immunogen (Remune; inactivated HIV-1 from which gp120 is depleted in incomplete Freund's adjuvant or adjuvant control at 0, 12, 24, and 36 weeks at five different clinical sites in Thailand. Neither group received antiviral drug therapy. The a priori primary endpoint for the trial was changes in CD4-cell counts with secondary parameters of percent changes in CD8-cell counts (percent CD4, CD8, and CD4/CD8) and body weight. Subsets of subjects were also examined for changes in plasma HIV-1 RNA levels, Western blot immunoreactivity, and HIV-1 delayed-type hypersensitivity (DTH) skin test reactivity. There was a significant difference in changes in CD4-cell counts that favored the HIV-1 immunogen-treated group compared to those for the adjuvant-treated control group ( P < 0.05). On average, for HIV-1 immunogen-treated subjects CD4-cell counts increased by 84 cells by week 40, whereas the increase for the control group was 38 cells by week 40. This increase in CD4-cell count was associated with increased HIV-specific immunogenicity, as shown by Western blotting and enhanced HIV-1 DTH skin reactivity. No significant differences in adverse events were observed between the groups. The results of this trial suggest that HIV-1 immunogen is safe and significantly increases CD4-cell counts and HIV-specific immunity compared to those achieved with the adjuvant control in asymptomatic HIV-1-infected subjects not taking antiviral drugs.

Seroprevalence of HHV-8 has been studied in Malaysia, India, Sri Lanka, Thailand, Trinidad, Jamaica and the USA, in both healthy individuals and those infected with HIV. Seroprevalence was found to be low in these countries in both the healthy and the HIV-infected populations. This correlates with the fact that hardly any AIDS-related Kaposi’s sarcoma has been reported in these countries. In contrast, the African countries of Ghana, Uganda and Zambia showed high seroprevalences in both healthy and HIV-infected populations. This suggests that human herpes virus-8 (HHV-8) may be either a recently introduced virus or one that has extremely low infectivity. Nasopharyngeal and oral carcinoma patients from Malaysia, Hong Kong and Sri Lanka who have very high EBV titres show that only 3/82 (3.7%) have antibody to HHV-8, demonstrating that there is little, if any, cross-reactivity between antibodies to these two gamma viruses.

55 Background: HHV-8 has been implicated in the pathogenesis of Kaposi's sarcoma (KS) as well as body cavity B-cell lymphoma based on PCR analysis of tissues and antibody to viral latent and late proteins. Materials & Methods: We developed an IgG antibody ELISA to HHV-8 using whole viral lysate which contained on average 1.9 × 109 viral particles/ml as well as >1.08 mg/ml of protein. An IFA to HHV-8 lytic antigen was made using the KS-1 cell line. In addition, Western blots to structural virus proteins were developed to elucidate immunodominant proteins that may be detected in sera. Results: Our serological data showed that approximately 11% of the healthy donors in the U.S.A. contained IgG antibody to HHV-8 compared to 12% in Thailand, 4% in India, and 15% in Malaysia (ethnic Chinese, Malays, and Bidayuhians). The antibody titer in healthy donors' sera by IFA ranged 1:20 - 1:320 compared to 1:320 - >1:10,000 in classical KS sera. Similar titers were evident by ELISA, and these assays correlated (p <0.0001, r=0.915). Approximately 50% of HIV-1 (+) homosexuals and >40% HIV-1 (+) IV drug users have HHV-8 serum antibody which suggests being at higher risk for KS. Sera from 93% of KS patients both HIV (+) and HIV (−) contained elevated IgG antibody as detected by IFA and ELISA. Body cavity B-cell lymphoma is the only other disease found associated with increased HHV-8 antibody levels. No evidence of HHV-8 antibody was evident in sera from multiple myeloma, Hodgkin's disease, acute and chronic leukemia, and other B- and T-cell lymphomas from the U.S. population. PCR in situ hybridization for HHV-8 DNA corroborated the disease association results found with the antibody tests. Conclusion: Our data showed variations in infection of HHV-8 in U.S.A. vs. India, Malaysia, and Thailand. Although KS has not been reported in Southeast Asia, HHV-8 infection may be linked to other diseases there. Our ELISA contains a majority of the viral structural proteins and, therefore, detects broad-spectrum antibodies which may be missed by other assays. Our IFA is directed against lytic HHV-8 antigens and correlates well with the ELISA assay. Therefore, these assays may be useful tools in large-scale seroepidemiological studies and clinical follow-up for at-risk individuals. Our findings indicate that the prevalence of HHV-8 is extremely low regardless of the geographic location suggesting that HHV-8 is an important human pathogen associated with KS or other unknown diseases compared to other human gamma, beta, and alpha herpesviruses.

The safety and immunogenicity of REMUNE, an HIV-specific immune based therapy for HIV infection, was evaluated in a cohort of 30 HIV infected subjects in Thailand. This therapy utilizes a gp120 depleted inactivated virus (HZ321), which exhibits a high degree of conservation with the core antigens of both type B' and E strains of HIV, the predominant Thailand isolates. The treatment was well tolerated, with no serious adverse events reported over the course of the 4-month trial. Treatment in which four doses were administered with REMUNE appeared to boost HIV-specific immune responses, with approximately 75% of the treated subjects demonstrating an increase in either the repertoire or the intensity of the serological response to HIV as measured by Western blot. CD4%, viral load, and weight remained stable over the course of the 4-month study relative to baseline values. Viral subtyping of this cohort revealed a predominance of type 'E'. These data suggest that REMUNE is safe and immunogenic in seropositive Thai subjects and supports further study of the therapeutic potential of REMUNE to treat HIV-1 infection.

Objective:To examine the effect of treatment with an inactivated, gp120-depleted, HIV-1 immunogen (Remune) in 30 Thai subjects infected with HIV-1 subtype E. Design:Sixty-week open-label study. Methods:Thirty HIV-positive volunteers with CD4 cell counts ≥ 300 × 106/l were given intramuscular injections of Remune into the triceps muscle on day 1 and then at weeks 4, 8, 12, 24, 36, 48 and 60. Results:Treatment with Remune was well-tolerated and augmented HIV-1-specific immune responses. Furthermore, subjects had a significant increase in CD4 cell count (P < 0.0001), CD4 cell percentage (P < 0.0001), CD8 cell percentage (P < 0.0001), and body weight (P < 0.0001) compared with pretreatment levels. Fourteen subjects with detectable viral load at day 1 showed a decrease at week 60 (P = 0.04). Retrospective Western blot analysis showed 23 subjects with increased intensity of antibody bands and 15 patients showed development of new reactivities to HIV proteins, especially towards p17 and p15. Conclusion:These results indicate that HIV-specific immune-based therapeutic approaches such as Remune should be further examined in countries with different clades of HIV-1 and where access to antiviral drug therapies is limited.

Antibodies against dengue viral proteins were demonstrated in sera from dengue-infected patients by polyacrylamide gel electrophoresis, Western blotting, and enzyme immunoassay. Primary dengue cases showed low titers of IgG class antibodies to envelope (E) proteins and two non-structural proteins, NS3 and NS5, in sera collected during the convalescent phase. Secondary dengue-infected patients always demonstrated IgG antibodies to E proteins in sera collected during the acute phase, and high titers of IgG antibodies to many other proteins, including NS1, NS3, NS5, and C proteins in sera collected during the convalescent phase. Appearance of antibodies to E, NS3, and NS5 could be detected within five days after the onset of fever. These three dengue viral proteins and their corresponding antibodies may be involved in the immunopathologic mechanism underlying this disease. For diagnostic purposes, identifying the non-structural proteins such as NS3 and NS5 may be the best means for early confirmation of the disease.

The suckling mouse which is used in the classical method to detect and propagate dengue viruses was evaluated in conjunction with the western blot and immunoenzymatic methods to detect the infecting strains of dengue viruses. After intracerebral inoculation of patients' sera into the suckling mice for 7 days, the mice were examined for the presence of dengue proteins, even though the mice did not have the neurological symptoms which usually serve as an indicator for the presence of dengue infection in the mouse brain. With a blind study of a set of 12 specimens, the suckling mice could detect the virus with the same frequency as the mosquito system but in shorter time of incubation period. The whole process to identify the type of infection takes 9 days. Another important finding is the demonstration of the virion antigen in the liver. The quantity and quality of viral proteins in liver are comparable to those in the brain suggesting that the virus may replicate in the liver as well as in the brain.

Antisera to all types of dengue and Japanese encephalitis (JE) viruses were raised in rabbits. The first set of rabbits was immunized with crude antigens prepared by a sucrose acetone extraction. The first generation of antisera demonstrated antibody activity towards the group specific flaviviruses, without unwanted antibody activity towards the mouse brain material. Antibody activity towards E, NS3, NS5 and NS1 could be observed by western blot/immunoenzymatic assay. Another set of rabbits was immunized with the precipitin complexes formed between antisera raised against each type of dengue, or JE viruses, and their homologous antigens. Each rabbit serum was screened again by the western blot/immunoenzymatic method, and was absorbed with other types of dengue viruses until the specific activity towards the immunized viral antigen was obtained. The last 2 bands detected on the viral antigen strip were the doublet of protein bands at Mr 67 and 71 kDa, which are the NS3 protein. The specific polyclonal antisera obtained can be used with other tests as well as the monoclonal antibody. Since absorption can lead to type specific antisera, this NS3 protein must be quite unique, it processes a type specific determinant (s) and deserves further study as a target molecule at the polypeptide level as well as at the RNA level.

The authors applied polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), western blot, and enzymeimmunoassay (EIA) for the detection of antibodies toward individual dengue viral proteins or polypeptides. SDS-PAGE procedure as described by Laemmli et al., was applied and modified. The results can be observed by visualization. The scores, from 0 to 4, can be assigned by comparing the intensity of the color development. Besides being sensitive and rapid, this technique yields information of the polypeptides or molecular level. An increasing of intensities of positive reactions indicated rising in antibodies titers and all serotypes of dengue viruses (from type 1 to type 4) can be tested together allowing reliable comparison among serotypes. With hyperimmune human sera, at least 13 polypeptides reacted with sera while negative non-immune subject showed no reaction. It is highly possible to use this technique as a rapid quantitative and for qualitative analysis of antibodies to individual viral proteins as well.

Peroxidase-antiperoxidase (PAP) staining was applied to measure the antibody enhancement activity in human monocytes. Increasing in number of infected cells can be seen with increasing of staining intensity of the cells by ordinary light microscope. Shifting of the optimum enhancement activity was found in previously tritiated antiserum indicated that for titration of antibody enhancement activity several dilutions of antiserum should be included in each experiment. Validity of the PAP method was made by the comparison of the results with Infectious Center Assay (ICA). With this technique, titration for antibody enhancement for dengue virus infection can be done with non-expensive equipment and can be kept for comparison for months.

Crossed immunoelectrophoresis of dengue type 2 virus revealed at least two precipitating antigens which shared some antigenic determinants. Glycoprotein components of both antigens were detected by binding to concanavalin A. Sera from dengue hemorrhagic fever patients showed precipitating antibodies to both antigens which could be quantitated according to the precipitate patterns formed in the intermediate gel of crossed immunoelectrophoresis. All secondary dengue hemorrhagic fever patients demonstrated an increase in precipitin titers in convalescence sera. Most patients with mild illness contained precipitating antibodies in acute phase sera whereas severe cases did not. Convalescent sera from severe cases showed only low titers. These precipitating antibodies may be associated with protection since they were produced early only in those with mild form of illness.

The unlabelled immunoperoxidase, peroxidase-anti-peroxidase (PAP), technique was used to detect dengue type-2 viral antigen in several cell systems including the endogenous peroxidase containing cells. These cells are the mosquito cell line (C6/36), continuous cell line of rhesus monkey kidney (LLC-MK2), human monocyte culture both cell suspension and monolayer, and human peripheral blood leukocytes. All of these specimens gave the same results that dengue-2 viral antigen presented in cytoplasm only and the patterns of marker presentation in positive cells varied depending on the duration after infection. The sensitivity of this method is extremely high since it can detect dengue-2 antigen after its attachment on mosquito cells (15 min) as seen in experiments with mosquito cell line, C6/36. False positive was not observed in all cell systems tested.

The ability of dengue viral antigen(s) from different sources, and performed antigen-antibody complexes, to activate the third component of human complement (C3) in vitro was demonstrated by using split products of human C3. Dengue type 2 viral antigen(s) from suckling mouse brain and from infected monocytes activated C3 via the alternative pathway, while performed soluble dengue-2 antibody complex activated mainly the classical pathway. The implications of this finding for the pathogenesis of dengue hemorrhagic fever are discussed.