Production and immunogenicity of hypoallergenic codon-optimized DNA vaccine encoding mature Der p 1 allergen.

BACKGROUND: Genetic vaccination with plasmid DNA encoding allergens is a promising potential approach for the treatment or prevention of allergy. Nonetheless, because the allergens expressed can display immunoglobulin (Ig) E reactivity, methods to deliver hypoallergenic variants can minimize the risk of type 2 helper (T(H)2) cell priming after DNA immunization. METHODS: A humanized synthetic gene encoding mature Dermatophagoides pteronyssinus group 1 (Der p 1) allergen was cloned into the pHIS expression vector carrying unmethylated CpG 2006 (CpG 2006) motif but devoid of signal sequence. The immunogenicity of this DNA construct was compared in naïve mice with that of recombinant ProDer p 1 protein adjuvanted with alum. RESULTS: Codon optimization of the cDNA encoding mature Der p 1 markedly improved allergen expression. Mature Der p 1, expressed intracellularly in Human Embryonic Kidney 293 cells (HEK 293 cells) transfected with codon-optimized Der p 1 cDNA (pHIS-mHuDer p 1), was shown to be hypoallergenic as it displayed no IgE reactivity. Intradermal vaccinations of naïve Balb/C mice with pHIS-mHuDer p 1 elicited an allergen-specific T(H)1 response characterized by the production of specific IgG2a, a very low amount of specific IgG1, and no specific IgE. Lipoplex formulation with cationic liposome composed of lecithin, N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) and cholesterol not only accelerated the induction of T(H)1 response but also increased its intensity. CONCLUSION: A codon-optimized DNA vaccine encoding mature Der p 1 in a lipoplex formulation could represent a promising hypoallergenic vaccine candidate for safer immunotherapy against house dust mite allergy.

Bayesian network analysis of resistance pathways against HIV-1 protease inhibitors.

Interpretation of Human Immunodeficiency Virus 1 (HIV-1) genotypic drug resistance is still a major challenge in the follow-up of antiviral therapy in infected patients. Because of the high degree of HIV-1 natural variation, complex interactions and stochastic behaviour of evolution, the role of resistance mutations is in many cases not well understood. Using Bayesian network learning of HIV-1 sequence data from diverse subtypes (A, B, C, F and G), we could determine the specific role of many resistance mutations against the protease inhibitors (PIs) nelfinavir (NFV), indinavir (IDV), and saquinavir (SQV). Such networks visualize relationships between treatment, selection of resistance mutations and presence of polymorphisms in a graphical way. The analysis identified 30N, 88S, and 90M for nelfinavir, 90M for saquinavir, and 82A/T and 46I/L for indinavir as most probable major resistance mutations. Moreover we found striking similarities for the role of many mutations against all of these drugs. For example, for all three inhibitors, we found that the novel mutation 89I was minor and associated with mutations at positions 90 and 71. Bayesian network learning provides an autonomous method to gain insight in the role of resistance mutations and the influence of HIV-1 natural variation. We successfully applied the method to three protease inhibitors. The analysis shows differences with current knowledge especially concerning resistance development in several non-B subtypes.

Survey of reverse transcriptase from the heterosexual epidemic of human immunodeficiency virus type 1 CRF01_AE in Thailand from 1990 to 2000.

Genetic diversity of the HIV-1 envelope gene has shown a steady increase over time in the Thai and other regional epidemics. A serial survey of subtype CRF01_AE polymerase gene (RT) diversity in Thailand was performed, using 48 novel and 15 reported sequences covering the period 1990--2000. These sequences were gathered from individuals whose sole risk factor for infection was heterosexual contact. By contrast to envelope, diversity was low and, despite a 40% increase early in the epidemic, has remained static since 1996. These results indicate that epidemic HIV-1 may be constrained within defined limits of genetic diversity at least in some genomic regions.

A randomized, double-blind trial of half versus standard dose of zidovudine plus zalcitabine in Thai HIV-1-infected patients (study HIV-NAT 001). HIV Netherlands Australia Thailand Research Collaboration.

BACKGROUND: Triple combination antiretroviral therapy, recommended as standard of care, is unaffordable for much of the developing world. OBJECTIVES: To establish whether half doses of zidovudine (AZT) and zalcitabine (ddC) are as effective as standard doses in a Thai population with lower body weight than Western populations and predominantly infected with HIV-1 subtype E. METHODS: A group of 116 antiretroviral naive patients, with CD4 cell counts 100-500 x 10(6) cells/l, were randomized to: AZT 200 mg three times daily plus ddC 0.75 mg three times daily versus AZT 100 mg three times daily plus ddC 0.375 mg three times daily and followed-up regularly for 48 weeks. RESULTS: The study enrolled 111 patients: 59 men and 52 women, body weight (mean +/- standard deviation) 56.4 +/- 12.3 kg, mean CD4 cell count 324 x 10(6) cells/l, mean HIV RNA 4.7 log10 copies/ml. There were no significant differences between the two groups. Twelve patients discontinued, including two deaths that were unrelated to study medication. No significant differences in adverse events were seen. Week 48 data for the standard dose and half dose arms, respectively, were mean CD4 cell count increases of 52 and 78 x 10(6) cells/l (P = 0.34), mean plasma HIV-1 RNA reduction of 1.4 and 1.1 log10 copies/ml (P = 0.10), HIV RNA of < 400 copies/ml in 52 and 20%[ (P = 0.001). Participants with higher than mean baseline CD8 cell counts (mean 1062 x 10(6) cells/l) showed greater decline in CD8 cells on standard doses. Further analysis showed improved reduction in HIV RNA (P < 0.0001) and in the percentage with undetectable HIV RNA (P = 0.0137) in the standard dose arm, corrected for baseline HIV RNA, which if < 4.75 log10 copies/ml significantly correlated with HIV RNA < 400 copies/ml at week 48. CONCLUSION: At week 48, the proportion with HIV RNA < 400 copies/ml was significantly higher in the standard dose arm; lower baseline HIV RNA correlated with better HIV RNA outcome at 48 weeks. The arms did not differ in CD4 cell response but standard doses correlated with greater CD8 cell decline.

A comparison of two dosing regimens of zidovudine in Thai adults with early symptomatic HIV infection. Conducting clinical HIV trials in South-East Asia.

AIM: To compare the clinical and immunological efficacy, and tolerance of two dosage regimens of zidovudine (ZDV) in an adult Thai population with early symptomatic human immunodeficiency virus (HIV) disease and to identify important clinical issues associated with conducting HIV trials in South-East Asia. METHODS: HIV-infected Thai adults, with early symptomatic HIV disease and CD4 lymphocyte counts less than 400/mm3, who were managed in the infectious diseases clinics at two university teaching hospitals in Bangkok, Thailand, were enrolled in a randomised, open-label, dose-regimen comparison trial of ZDV. Two oral ZDV dosing regimens: regimen A, 100 mg tid+200 mg nocte (ZDV-A) vs regimen B, 250 mg bid (ZDV-B) were compared. The main outcome measures were: 1. Clinical efficacy: rate of progression to acquired immunodeficiency syndrome (AIDS) or death. 2. Immunologic efficacy: changes in CD4 lymphocyte numbers compared to baseline; rate of decline of CD4 lymphocyte numbers to less than 100/mm3. 3. Toxicity, as defined by clinical symptomatology and laboratory parameters. RESULTS: Two hundred and four patients were enrolled (103 ZDV-A; 101 ZDV-B) of whom 195 were followed beyond baseline. Patients were typical of those encountered with HIV in Thailand: mean age 33 years; 89% male; 88% heterosexual HIV acquisition; mean baseline CD4 lymphocyte count 241/mm3. Follow-up while on therapy was comparable for the two groups (mean+/-SD): 533+/-236 days (ZDV-A) vs 592+/-210 days (ZDV-B). One hundred and eleven patients (57%; 51 ZDV-A; 60 ZDV-B) were treated for at least 22 months (669+/-30 days). Clinical and immunological outcomes for ZDV-A and ZDV-B, including rate of progression to AIDS or death, development of non-AIDS-defining opportunistic infections, mean changes in CD4 lymphocyte numbers/mm3, difference in area under the CD4:time distribution curve and difference in the rate of decline of CD4 lymphocyte numbers to less than 100/mm3, were not significantly different. The presence of oral hairy leukoplakia or unintentional weight loss of 10-20% at enrollment were significantly associated with the later development of AIDS (p=0.03 and 0.04, respectively). ZDV-associated toxicity was similar for both regimens. Maintaining protocol adherence and appropriate clinical follow-up emerged as important practical issues. CONCLUSION: In Thai adults, ZDV 100 mg tid+200 mg nocte and ZDV 250 mg bid have similar clinical and immunological efficacy. Rates of ZDV toxicity are comparable to those reported in non-Asian populations. Despite limitations in medical care access and maintaining long-term follow-up, successful trials of antiretroviral agents are feasible in South-East Asia and multi-drug treatment trials should be pursued in appropriate institutions.

Thai Red Cross zidovudine donation program to prevent vertical transmission of HIV: the effect of the modified ACTG 076 regimen.

OBJECTIVES: To evaluate the impact of the modified ACTG 076 zidovudine regimen on the risk for vertical HIV transmission. DESIGN: Observational retrospective evaluation of a prospective cohort. SETTING: Thai Red Cross zidovudine donation program to reduce vertical HIV transmission. PATIENTS: HIV-infected Thai women and their offspring. INTERVENTION: The modified regimen consisted of 500 mg zidovudine daily during pregnancy and 300 mg zidovudine every 3 h during labor, taken orally, and 2 mg/kg zidovudine syrup four times daily for 6 weeks to infants. MAIN OUTCOME MEASURES: Only infants with at least 1 HIV DNA polymerase chain reaction (PCR) result at age > or = 4 weeks were included. HIV infection was defined by having at least one positive PCR at age > or = 4 weeks. The transmission rate was calculated. Characteristics of women who did and did not transmit HIV to infants were compared. RESULTS: A total of 2891 women and their infants participated in the program and 726 infants of 719 women were included in the analysis. Forty-three infants were infected. The overall transmission rate was 6.0% (95% confidence interval, 4.4-8.0). There were no differences in maternal characteristics between transmitters and non-transmitters. The transmission rate in women who started zidovudine before 30 weeks' gestation was not significantly different from that in women who started zidovudine at or after 30 weeks' gestation: 5.7 versus 3.3%, respectively. CONCLUSIONS: This modified zidovudine regimen is effective in reducing vertical transmission in a country with predominant subtype E infection. A donation program for preventing vertical HIV transmission can be implemented in developing countries, as in Thailand.

Clinical and immunological features of human immunodeficiency virus infection in patients from Bangkok, Thailand.

BACKGROUND: To assess the association between the CD4 count and clinical diseases in a cohort of Thai patients. METHODS: In all, 1902 patients who presented with human immunodeficiency virus (HIV) infection at the Chulalongkorn University Hospital in Bangkok were investigated. RESULTS: At the time of presentation 295 (15.5%) patients had acquired immunodeficiency syndrome (AIDS) and there was a highly significant tendency for lower CD4 counts in this group (median 67/mm3) than in patients free of AIDS (median 369/mm3). A total of 757 patients had data available on follow-up and were free of AIDS at the first visit. During a median follow-up of 0.9 years, 110 developed AIDS or AIDS-related death (12.2/100 person years). Subjects with CD4 count < 200/mm3 at initial visit showed over a ninefold increase in risk of developing AIDS compared to subjects with levels > or = 500/mm3 (relative risk [RR] = 9.1; 95% CI: 5.4-16.0). The rate/100 person years was 47.1 compared with 6.0 in subjects with levels > or = 500/mm3. After adjusting for initial CD4 count, homosexual men showed over a twofold increase in risk of developing AIDS compared to heterosexuals (RR = 2.4; 95% CI: 1.6-4.4) and intravenous drug users (IVDU) showed nearly a twofold increase (RR = 1.8; 95% CI: 0.9-3.9). The increased risk in homosexual men persisted even after further adjustment for clinical stage (RR = 2.2; 95% CI: 1.3-3.7) but the increased risk in IVDU was attenuated (RR = 1.5; 95% CI: 0.7-3.2) although it remained increased albeit non-significantly. Men tended to progress faster to AIDS than women but the difference was not significant. However, the faster progression in homosexual men was seen even when compared to heterosexual men only. CONCLUSION: The rate of progression of AIDS according to CD4 count group at baseline in this Thai cohort is broadly comparable with Western cohorts. It appears that heterosexuals in Thailand show slower progression to AIDS than homosexual men.

PIP: The natural history of HIV infection was investigated in a cohort of 1902 HIV-positive patients (median age, 29 years) seen at the Chulalongkorn University Hospital in Bangkok, Thailand, in 1985-90 in whom a CD4 count was performed at the initial visit. The majority (64%) were male heterosexuals; 10% were homosexual men, 10% reported intravenous drug use, and 16% were heterosexual women. At the time of study enrollment, 295 patients (15.5%) had progressed to AIDS. AIDS patients had a significantly lower CD4 count (median, 67/cu. mm) than those without AIDS (median, 369/cu. mm). Of the 757 patients who were AIDS-free at baseline and available for follow up (median time, 0.9 years), 110 developed AIDS or died from an AIDS-related cause (12.2/100 person-years). Tuberculosis was the major AIDS-defining illness in all risk groups. 50% of those with a CD4 count under 200/cu. mm at the initial visit developed AIDS within 2 years compared with 12% of those with levels of 500/cu. mm (relative risk (RR), 9.1; 95% confidence interval (CI), 5.4-16.0). The rates per 100 person-years were 47.1 and 6.0, respectively. After adjusting for initial CD4 count, homosexual men had a relative risk of developing AIDS of 2.4 (95% CI, 1.6-4.4) compared with heterosexuals; intravenous drug users had a relative risk of 1.8 (95% CI, 0.9-3.9). The increased risk in homosexual men persisted even after further adjustment for clinical stage (relative risk, 2.2; 95% CI, 1.3-3.7), but the increased risk in intravenous drug users was attenuated (RR, 1.5; 95% CI, 0.7-3.2). Older patients progressed faster to AIDS than younger patients, but there was no significant difference between men and women in the course of disease. These findings are consistent with those of studies conducted in Western countries indicating a slower progression to AIDS in heterosexual men than homosexuals and a correlation between CD4 count and rate of progression to AIDS.

International clinical trials of HIV vaccines: I. Phase I trial of an HIV-1 synthetic peptide vaccine in Bangkok, Thailand.

A randomized, double blind, placebo controlled Phase I trial of a prototype human immunodeficiency virus type 1 (HIV-1) synthetic peptide vaccine was conducted in Bangkok, Thailand, to evaluate the safety and immunogenicity of the vaccine in a population of healthy adults at low risk for HIV infection, and to establish essential infrastructure for future HIV vaccine trials in Thailand. Thirty volunteers (25 males; 5 females) were recruited and randomized into 3 groups, receiving 3 intramuscular injections of either 100 micrograms vaccine (N = 12) or 500 micrograms vaccine (N = 12) or alum placebo (N = 6) on weeks 0, 4 and 25. The vaccine was well tolerated without any serious adverse effects. HIV-1 specific ELISA responses were detected in 20/24 subjects who received the vaccine, with V3 binding antibody titers ranging from 1:69 to 1:5,041. HIV-1 (MN) specific neutralizing antibody was detected in 19/20 of subjects with detectable HIV-1 specific binding antibody. Neutralization titers ranged from 1:14 to 1:1,294, which were less than titers observed in HIV-infected subjects. The results of this study indicate that the vaccine was well tolerated, and that the vaccine stimulated anti-HIV humoral immune responses in Thai subjects. The successful undertaking of this first HIV vaccine trial conducted in Thailand provided important preparatory information surrounding volunteer recruitment and motivations, and paves the way for future trials of HIV vaccines in Thailand.

Should travellers in rabies endemic areas receive pre-exposure rabies immunization?

A questionnaire survey was conducted in 1,882 foreign travellers, 74% of which were Europeans, after being in Thailand for an average of 17 days, about the history of potential rabies exposure during their visits. Dog bite and dog lick were experienced in 1.3% and 8.9% of the travellers respectively. The exposed individuals tended to stay in Thailand longer and the incidents occurred mainly in cities rather than in the rural areas. Thirty-one (1.6%) of all travellers had a history of rabies vaccination, 9 as a result of dog bite or dog lick in Thailand whereas the remaining 22 had already received the vaccine prior to coming to Thailand. Such high prevalences of potential rabies exposure and rabies vaccination may justify the inclusion of rabies vaccine into the multiple vaccination program for travellers to rabies endemic countries. This was favoured by over half of the travellers interviewed.

Evaluation of a 2-minute anti-human immunodeficiency virus (HIV) test using the autologous erythrocyte agglutination technique with populations differing in HIV prevalence.

A total of 1,800 blood specimens (1,000 from healthy blood donors, 300 from patients with sexually transmitted disease, and 500 from intravenous drug users) were simultaneously tested with anti-human immunodeficiency virus enzyme-linked immunosorbent assay (ELISA) kits and a newly developed 2-min test for anti-human immunodeficiency virus based on the principle of autologous erythrocyte agglutination (AGEN Biomedical Limited). We found that AGEN's rapid test was as sensitive and specific as the other ELISA kits.

Clinical correlation of the immunological markers of HIV infection in individuals from Thailand.

OBJECTIVE: To evaluate the usefulness of T-cell subsets, beta-microglobulin (B2M), p24 antigen and anti-p24 antibodies as differentiating and prognostic markers in HIV-infected Thai patients. DESIGN: Sixty-one HIV-infected patients in various stages of disease (six AIDS, three AIDS-related complex, 34 persistent generalized lymphadenopathy and 18 HIV-asymptomatic) were followed prospectively for 2 years. Patients were examined and immunological markers assessed every 6 months at least. Any HIV-related complications were treated symptomatically and clinical staging was re-evaluated at each visit. Due to financial constraints, none of the patients were given antiretroviral drugs. METHODS: T-cell subsets were enumerated by indirect immunofluorescence using OKT4 or OKT8 for T-helper and T-suppressor cells, respectively. beta 2M and p24 antigen were quantified by enzyme-linked immunosorbent assay and anti-p24 antibodies were by immunoblot assay. RESULTS: Our preliminary study revealed that the decrease in CD4+ T-cells or anti-p24 titre and the increase in p24 antigen or beta 2M correlated well with disease staging, as defined by the Centers for Disease Control. Absolute number and percentage of CD4+ T-cells, absolute number of CD8+ T-cells, beta 2M level and p24 antigen and anti-p24 antibody levels at entry could be used as reliable prognostic markers for HIV progression. The combination of p24 antigen with the number of CD4+ T-cells substantially increased the prognostic value, compared with either used alone. CONCLUSIONS: The annual rate of clinical progression from asymptomatic to symptomatic HIV infection in our study was 6.8%. The results we obtained in this preliminary study may be used as baseline data for planning future therapeutic interventions in Asian patients.

Comparison of the sensitivity of various anti-HIV tests in early seroconversion sera.

Paired sera from 4 patients with proven HIV infection whose initial specimens obtained 14-51 days earlier were indeterminate were simultaneously retested with 7 screening anti-HIV test kits and the immunoblot assay. The study aimed to evaluate the sensitivity of various new and old anti-HIV screening tests. The test kits evaluated were 4 ELISA test kits from Wellcome (Wellcozyme), Organon (Vironostika anti-HTLV-III), Pasteur (Rapid Elavia) and Diagnostic Biotechnology (DB, HIV-1 ELISA), 2 rapid tests based on microfiltration enzyme immunoassay procedure from Rapport (SUDS) and Disease Detection International (SeroCard), and 1 particle agglutination (PA) test (Serodia-HIV). Immunoblot strips from Diagnostic Biotechnology (HIV-1 Western blot) were used to confirm the HIV infection in these serum specimens. Out of the 4 initial serum specimens tested, all were positive by PA, 2 by SUDS, Wellcome and Pasteur, 1 by SeroCard and DB, and none by Organon. When tested by immunoblot, 1 was negative (i.e., completely without any bands) whereas 3 were indeterminate (i.e., 1 with very weak band for p18, 1 with weak band for p24, 1 with very weak band for gp160. All repeat specimens obtained 14-51 days later (mean 32.5 +/- 16 days) were positive by all screening tests as well as immunoblot. Therefore, with these 4 early seroconversion sera, the sensitivity of the PA was 100%, that of SUDS, Wellcome and pasteur was 50%, of that SeroCard and DB was 25%, and Organon, 0%. None of these sera was considered positive by immunoblot.

Serum antibodies to HTLV-I in Thai patients with chronic progressive myelopathy, multiple sclerosis, myopathy and in HIV-seropositive intravenous drug abusers.

Antibodies to HTLV-I were assayed in sera of 9 patients with progressive myelopathy, 11 with multiple sclerosis, 5 with myopathy and in 10 HIV-seropositive intravenous heroin abusers. Clinical features in 9 cases with progressive myelopathy were not different from those previously described in tropical spastic paraparesis associated with HTLV-I infection. No detectable HTLV-1 antibody was found in the sera of any of the 35 patients studied.

Comparative immunogenicity study of four plasma-derived hepatitis B vaccines in Thai young adults.

Immunogenicity of four plasma-derived hepatitis B vaccines (Merck, Sharp and Dohme, Pasteur, Dutch CLB and Korean Cheil-Sugar) was compared in Thai young adults. After primoimmunization, only the Merck and Pasteur vaccines could achieve greater than 90% seroconversion (i.e. anti-HBs greater than or equal to 10 mIU ml-1) whereas both the CLB and Korean vaccines needed a fourth dose to achieve this level of seroconversion. The anti-HBs titres of both heat-inactivated vaccines (CLB and Korean) were also significantly lower than those of the other two vaccines. We propose that the HBsAg content in both heat-inactivated vaccines should be increased and a booster (fourth) dose should be given in order to enhance their immunogenicities.

Humoral and cell-mediated immune responses to various economical regimens of purified Vero cell rabies vaccine.

Purified Vero cell rabies vaccine (PVRV) is a new effective but inexpensive tissue culture rabies vaccine for human use. We investigated if the cost of immunization with PVRV could be further reduced by intradermal immunization. Fifty-eight subjects with low-risk exposure to rabies were randomized into 4 groups to receive full-dose (0.5 ml) intramuscular injection of PVRV on days 0, 3, 7, 14 and 28 or 4, 2 or 1 intradermal injections of PVRV (0.1 ml) on days 0, 3, and 7, followed by another intradermal injection on day 28. Neutralizing antibodies and specific cell-mediated response (CMIR) were sequentially followed up to day 36. The antibody levels in the intradermal groups increased with the number of injection sites and the levels achieved by the 2-site i.d. regimen were not significantly different from those obtained by the full-dose i.m. even though only 1/3 of the amount of PVRV was used. Specific CMIR occurred 1 week sooner in the 2 and 4-site i.d. regimens than the full-dose i.m. We therefore recommended that our 2-site i.d. regimen of PVRV should be further tested with a view to substituting it for the more expensive full-dose i.m. regimen in order to further reduce the cost of rabies prophylaxis particularly in the developing countries.