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An abstract of the research paper "Antibody responses to V2 loop are induced by CRF01_A E and not Clade B envelopes," by N. Karasawas and colleagues is presented.

Sexual transmission is the principal driver of the human immunodeficiency virus (HIV) pandemic. Understanding HIV vaccine-induced immune responses at mucosal surfaces can generate hypotheses regarding mechanisms of protection, and may influence vaccine development. The RV144 (ClinicalTrials.gov NCT00223080) efficacy trial showed protection against HIV infections but mucosal samples were not collected, therefore, the contribution of mucosal antibodies to preventing HIV-1 acquisition is unknown. Here, we report the generation, magnitude and persistence of antibody responses to recombinant gp120 envelope and antigens including variable one and two loop scaffold antigens (gp70V1V2) previously shown to correlate with risk in RV144. We evaluated antibody responses to gp120 A244gD and gp70V1V2 92TH023 (both CRF01&#95;AE) and Case A2 (subtype B) in cervico-vaginal mucus (CVM), seminal plasma (SP) and rectal secretions (RS) from HIV-uninfected RV144 vaccine recipients, who were randomized to receive two late boosts of ALVAC-HIV/AIDSVAX®B/E, AIDSVAX®B/E, or ALVAC-HIV alone at 0 and 6 months. Late vaccine boosting increased IgG geometric mean titers (GMT) to gp120 A244gD in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM (28 and 17 fold, respectively), followed by SP and RS. IgG to gp70V1V2 92TH023 increased in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM (11-17 fold) and SP (2 fold) two weeks post first boost. IgG to Case A2 was only detected in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM. Mucosal IgG to gp120 A244gD (CVM, SP, RS), gp70V1V2 92TH023 (CVM, SP), and Case A2 (CVM) correlated with plasma IgG levels (p<0.001). Although the magnitude of IgG responses declined after boosting, anti-gp120 A244gD IgG responses in CVM persisted for 12 months post final vaccination. Further studies in localization, persistence and magnitude of envelope specific antibodies (IgG and dimeric IgA) in anogenital secretions will help determine their role in preventing mucosal HIV acquisition.

The RV144 prime-boost regimen demonstrated efficacy against HIV acquisition while VAX003 and VAX004 did not. Although these trials differed by risk groups, immunization regimens, and immunogens, antibody responses may have contributed to the differences observed in vaccine efficacy. We assessed HIV-specific IgG, both total and subclass, and IgA binding to HIV envelope (Env): gp120 proteins and Cyclic V2 (CycV2) and CycV3 peptides and gp70 V1 V2 scaffolds in these 3 HIV vaccine trials. After two protein immunizations, IgG responses to 92TH023 gp120 (contained in ALVAC-HIV vaccine) were significantly higher in RV144 but responses to other Env were higher in the VAX trials lacking ALVAC-HIV. IgG responses declined significantly between vaccinations. All trials induced antibodies to gp70 V1 V2 but VAX004 responses to 92TH023 gp70 V1 V2 were weak. All CycV2 responses were undetectable in VAX004 while 92TH023 gp70 V1 V2 was detected in both RV144 and VAX003 but MN CycV2 was detected only in VAX003. Multiple protein vaccinations in VAX trials did not improve magnitude or durability of V1 V2 and CycV2 antibodies. Herpes simplex virus glycoprotein D (gD) peptide at the N terminus of AIDSVAX ® B/E and B/B gp120 proteins induced antibodies in all trials, although significantly higher in VAX trials. gD peptide induced IgA, IgG1, IgG2, and IgG3 but not IgG4. Multiple protein vaccinations decreased IgG3 and increased IgG4 changing subclass contribution to total IgG. Although confounded by different modes of HIV transmission, higher Env-specific IgA and IgG4 binding antibodies induced in the VAX trials compared to RV144 raises the hypothesis that these differences may have contributed to different vaccine efficacy results.

Background: The RV144 ALVAC-HIV prime, AIDSVAX B/E boost afforded 60% efficacy against human immunodeficiency virus (HIV) acquisition at 1 year, waning to 31.2% after 3.5 years. We hypothesized that additional vaccinations might augment immune correlates of protection., Methods: In a randomized placebo-controlled double-blind study of 162 HIV-negative RV144 vaccine recipients, we evaluated 2 additional boosts, given 6-8 years since RV144 vaccination, for safety and immunogenicity, at weeks 0 and 24. Study groups 1-3 received ALVAC-HIV+AIDSVAX B/E, AIDSVAX B/E, and ALVAC-HIV, respectively, or placebo., Results: Vaccines were well tolerated. For groups 1 and 2, plasma immunoglobulin (Ig) G, IgA, and neutralizing antibody responses at week 2 were all significantly higher than 2 weeks after the last RV144 vaccination. IgG titers against glycoprotein (gp) 70V1V2 92TH023 increased 14-fold compared with 2 weeks after the last RV144 vaccination (14069 vs 999; P < .001). Groups 1 and 2 did not differ significantly from each other, whereas group 3 was similar to placebo recipients. Responses in groups 1 and 2 declined by week 24 but were boosted by the second vaccination, albeit at lower magnitude than for week 2., Conclusions: In RV144 vaccinees, AIDSVAX B/E with or without ALVAC-HIV 6-8 years after initial vaccination generated higher humoral responses than after RV144, but these responses were short-lived, and their magnitude did not increase with subsequent boost., Clinical Trials Registration: NCT01435135., (Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

RV144 correlates of risk analysis showed that IgG antibodies to gp70V1V2 scaffolds inversely correlated with risk of HIV acquisition. We investigated IgG antibody responses in RV135 and RV132, two ALVAC-HIV prime-boost vaccine trials conducted in Thailand prior to RV144. Both trials used ALVAC-HIV (vCP1521) at 0, 1, 3, and 6 months and HIV-1 gp120MNgD and gp120A244gD in alum (RV135) or gp120SF2 and gp120CM235 in MF59 (RV132) at 3 and 6 months. We assessed ELISA binding antibodies to the envelope proteins (Env) 92TH023, A244gD and MNgD, cyclicV2, and gp70V1V2 CaseA2 (subtype B) and 92TH023 (subtype CRF01&#95;AE), and Env-specific IgG1 and IgG3. Antibody responses to gp120 A244gD, MNgD, and gp70V1V2 92TH023 scaffold were significantly higher in RV135 than in RV132. Antibodies to gp70V1V2 CaseA2 were detected only in RV135 vaccine recipients and IgG1 and IgG3 antibody responses to A244gD were significantly higher in RV135. IgG binding to gp70V1V2 CaseA2 and CRF01&#95;AE scaffolds was higher with the AIDSVAX(®)B/E boost but both trials showed similar rates of antibody decline post-vaccination. MF59 did not result in higher IgG antibody responses compared to alum with the antigens tested. However, notable differences in the structure of the recombinant proteins and dosage used for immunizations may have contributed to the magnitude and specificity of IgG induced by the two trials.

Background: In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case-control analysis to identify antibody and cellular immune correlates of infection risk., Methods: In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up., Results: Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P=0.02; q=0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P=0.03; q=0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies., Conclusions: This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.

Immune responses to vaccines may be influenced or associated with allelic variants of host genes such as those encoding human leucocyte antigens (HLA). We have molecularly determined the HLA class II DR and DQ gene, allele and haploype profiles in HIV-1 negative ethnic Thai recipients of an HIV-1 prime boost vaccine regimen, designed to induce neutralizing antibody (NAb) responses to HIV-1 CRF01&#95;AE. Non-response to vaccine associated with DRB1*11 (3/32 responders vs. 7/13 non-responders, p(c)=0.027) and DRB1*16:02 (0/32 responders vs. 4/13 non-responders, p(c)=0.078) alleles. Furthermore, vaccine recipients with HLA-DQ heterodimers encoded by DQA1*05:01 and DQB1*03:01 alleles, were much less likely to produce NAb (p=0.009). These data suggest that the lack of response to a vaccine designed to induce clade-specific NAb to HIV-1 is associated with the presence of certain HLA class II alleles and heterodimers in some Southeast Asians., (Copyright © 2011. Published by Elsevier Ltd.)

A phase I randomized, double blind, placebo-controlled trial to assess the immunogenicity of a multiclade HIV-1 DNA plasmid vaccine was conducted in 31 HIV-1-negative Ugandans. Following immunization with DNA at 0, 1, and 2 months, the frequency of HIV-specific immune responses was assessed up to 10 months using a standard chromium release assay (CRA), lymphoproliferative assay (LPA), and antibody dependent cell-mediated cytotoxicity assay (ADCC). Seven of 15 (47%) vaccinees demonstrated CTL activity using the CRA to HIV-1 Env B with responses observed 1 month following the second vaccination and as late as 7 months following complete immunization. Additionally, lymphoproliferative reponses were observed in 14/15 vaccinees against p24. No CTL or LPA responses were observed at baseline or in the placebo group. ADCC activity was minimally induced by DNA vaccination. This study demonstrates that immunization with DNA alone induces CTL and lymphoproliferative responses in a population that will participate in a phase IIb study evaluating HIV-1 DNA priming followed by boosting with a replication-defective recombinant adenovirus vector.

Background: The development of an effective HIV-1 vaccine is critical to control the pandemic. A prime-boost HIV-1 vaccine trial assessing safety and immunogenicity was conducted in Thailand as part of an evaluation of candidate regimens for a phase 3 efficacy trial., Methods: ALVAC-HIV (vCP1521), expressing circulating recombinant form 01&#95;AE (CRF01&#95;AE) gp120/subtype B LAI and subtype B Gag/Protease boosted with recombinant envelope oligomeric CRF01&#95;AE gp160 (ogp160) or bivalent CRF01&#95;AE/subtype B gp120 CM235/SF2, was evaluated in a phase 1/II trial of 130 HIV-negative Thai adults., Results: One hundred forty volunteers were enrolled, and 130 completed all safety and immunogenicity visits. Reactogenicity was common but generally mild, and there was no significant difference in the adverse event rate between vaccine and placebo recipients (P = 0.26). There were 7 serious adverse events during the follow-up period, none of which were vaccine related. Cumulative HIV-specific, CD8-mediated, cytotoxic T-lymphocyte responses were observed in 11 (25%) of 44 subjects who received ALVAC boosted by bivalent gp120 and in 5 (11%) of 45 subjects who received ALVAC boosted by ogp160, but these differences were not statistically significant compared with those in placebo recipients (P = 0.62 and P = 0.37, respectively). HIV-specific lymphoproliferative responses were detected in 84% of subunit-boosted vaccine recipients and in 10% of placebo recipients. Neutralizing antibody responses to CRF01&#95;AE and subtype B laboratory strains were seen in 95% of ogp160-boosted and 100% of gp120 B/E-boosted vaccinees, respectively., Conclusions: These 2 different prime-boost regimens seem to be safe and displayed cell-mediated immune responses consistent with those in other trials of canarypox vectors.

Antibody-dependent cell-mediated cytotoxicity (ADCC) was assessed in volunteers participating in an ALVAC-HIV (vCP1521)/AIDSVAX B/E gp120 prime-boost vaccine trial in Thailand. ADCC activity was measured using chromium release from gp120 subtype B- and CRF01&#95;AE-coated targets in 95 vaccinees and 28 placebo recipients. There was a significant difference in the magnitude of the ADCC response to both targets between vaccinees and placebo recipients. The frequency of responders to subtype B and to CRF01&#95;AE was 96% and 84% in the vaccine group versus 11% and 7% in the placebo group. The results demonstrate that this HIV vaccine is a potent inducer of ADCC activity and may be an additional protection of this prime-boost vaccine in preventing HIV disease.

ALVAC-HIV (vCP1521) and AIDSVAX B/E were evaluated in a phase 1/2 trial of human immunodeficiency virus (HIV)-negative Thai adults. Of 133 volunteers enrolled, 122 completed the trial. There were no serious vaccine-related adverse events, nor were there intercurrent HIV infections. Lymphoproliferative responses to glycoprotein 120 E were induced in 63% of the volunteers, and HIV-specific CD8 cytotoxic T lymphocyte responses were induced in 24%. Antibody responses increased in frequency and magnitude in association with the dose level of AIDSVAX B/E. Binding and neutralizing antibodies to the MN strain were induced in 100% and 98%, respectively, of the volunteers receiving 600 microg of AIDSVAX B/E, and such antibodies to E strains were induced in 96% and 71%, respectively, of these volunteers. This vaccine combination was well tolerated and was immunogenic, meeting milestones for advancement to phase 3 evaluation.

A phase I/II prime-boost vaccine trial in HIV-1-seronegative adults was conducted in Thailand using ALVAC-HIV (vCP1521) as a prime, boosting with either oligomeric gp160 TH023/LAI or Chiron HIV Thai subtype E (CM235) plus U. S. subtype B (SF2) gp120. Cytotoxic T lymphocyte (CTL) assays were conducted at one of the vaccine trial sites (Siriraj Hospital) at a single time point following the completion of immunization demonstrated that 8 of 50 (16%) vaccine recipients showed HIV-specific CTL by standard chromium release assay (CRA) after in vitro stimulation (IVS) for 2 weeks. Five additional vaccinees (13/50 = 26%) showed CTL responses after IVS for up to 4 weeks. Moreover, one volunteer with a positive CTL response to a single HIV antigen at Day 14 demonstrated a response to an additional HIV-1 antigen(s) after the longer IVS period. CTL activity was CD8+ restricted. Despite extension of the IVS up to 4 weeks, no CTL responses were detected in placebo recipients. These results imply that extension of the IVS period may increase the sensitivity of the CRA when measuring HIV-specific CTL in ALVAC-HIV prime-boost recipients without compromising specificity.

The inability to distinguish recrudescent Plasmodium falciparum infections (treatment failures) from reinfections (new infections) is an important impediment to the evaluation of antimalarial treatment regimens. Ten paired primary and recrudescent isolates collected near the Thai-Cambodian border were analyzed by restriction fragment length polymorphism (RFLP) and by polymerase chain reaction (PCR) genotyping of the genes encoding the following proteins: circumsporozite (CS) protein, erythrocyte binding antigen (EBA)-175, ring-infected erythrocyte surface antigen (RESA), merozoite surface protein-1 (MSP-1), and MSP-2. Both methods demonstrated that the fingerprint pattern of each recrudescent isolate was identical to or was contained within the pattern of the primary isolate. Each recrudescent isolate was unique when compared with the other nine primary isolates. Typing by PCR was more sensitive for the detection of multiclone infections and could be performed with small volumes of whole blood. The PCR genotyping could be a practical method for distinguishing a recrudescent from a new infection when treatment studies are conducted in areas with active malaria transmission.

Malaria parasites circulating in the blood or developing in the mosquito host can easily be seen and studied. We know much less about malaria parasites in the liver not only because of their location, but also because there are so few of them. Hepatic parasites are most often grown within hepatoma cells in culture, stained and visualized on slides under direct microscopy. In this report, Chitraporn Karnasuta and George Watt investigate the potential of cytocentrifugation as a tool for improving the detection of liver-stage malaria parasites.

Plasmodium falciparum parasites develop in the liver before being released into the bloodstream, where they exert the potentially lethal effects characteristic of malaria. Our understanding of the hepatic phase of the life cycle is limited by the parasite's requirement for fresh human liver cells in which to mature. In this work, liver parasites completed their development within a Thai human hepatoma cell line (HHS-102), and the presence of ring-form parasites in erythrocytes overlying the liver cell culture confirmed that an entire liver cycle was completed, culminating in the production of viable blood-stage parasites. The HHS-102 cell line allows investigation of the undefined liver stage of falciparum malaria previously unavailable in the laboratory.

Crude antigens prepared from the infective stage larvae of Trichinella spiralis were used for antibody detection by indirect ELISA and Western blotting in serum samples taken from trichinellosis patients and from normal, parasite-free controls. The serum specimens were collected from acute ill, symptomatic patients on the first day of treatment (Day 0), and then two months (M2) and 4 months (M4) later. The sensitivities of the indirect ELISA and Western blotting on Day 0 were 81% and 92%, respectively. Both tests were 100% sensitive for M2 and M4 serum samples. Every serum sample from the parasite-free controls tested negative by both immunological assays, indicating 100% specificity. Crude somatic antigens can therefore be used for the early detection of human trichinellosis (acute trichinellosis).

New treatments for malaria are urgently needed in areas such as Thailand where highly drug-resistant strains of Plasmodium falciparum are prevalent. Mefloquine is rapidly losing efficacy and conventional doses of halofantrine are infective. We therefore used pharmacokinetic stimulation to design an extended-dose halofantrine regimen and tested it in 26 soldiers stationed along the Thai-Cambodian border. Halofantrine was given after meals as three doses of 500 mg each at 4-hr intervals on the first day, followed by 500 mg a day for six days (total dose 4.5 g). Twenty-six soldiers treated with quinine-tetracycline for seven days (Q7T7) served as controls. There were no significant differences in efficacy between halofantrine and Q7T7 (P > 0.1) as assessed by cure rate (92% versus 85%), mean parasite clearance time (82 hr versus 81 hr), or mean fever clearance time (93 hr versus 99 hr). Halofantrine was better tolerated than Q7T7. The side effects score was lower (2 versus 11; P < 0.001), there were less days on which side effects occurred (2.0 days versus 5.5 days; P < 0.001), and fewer patients had adverse effects on every treatment day (4% versus 42%; P < 0.01). High-dose halofantrine is as effective and better tolerated than quinine-tetracycline for multidrug-resistant falciparum malaria.

Different lineages of hepatocellular carcinoma (HCC) were identified by the application of selected monoclonal antibodies to the study of the sequential histopathological changes which occurred during two regimens of chemical carcinogenesis in the rat. One regimen, that of Solt-Farber, caused prominent oval cell proliferation and large multiple neoplastic nodules, and the other regimen, continuous administration of diethylnitrosamine, produced minimal oval cell proliferation and a few small nodules. However, both regimens produce HCC in most exposed rats. Three monoclonal antibodies to liver cells, OV-6, H-4, and T-6, were selected on the basis of different tissue staining. OV-6 stains the cytoskeleton of bile duct cells, oval cells, and HCC but not that of hepatocytes. H-4 stains the cytoplasm of hepatocytes but of not hepatomas. T-6 stains the cytoskeleton of HCC only. In the Solt-Farber model, the monoclonal antibodies identified groups of hepatocytes within the persistent neoplastic nodules which had acquired the OV-6 epitope and had lost the H-4 epitope. HCC derived from this regimen had the same staining pattern, suggesting that the OV-6 positive H-4 negative hepatocytes were the precursors of the HCC. The presence within the nodules of oval cells, atypical duct structures, cells intermediate between duct cells and hepatocytes, and nodular hepatocytes all containing the OV-6 epitope raises the possibility that any of these cell types could serve as the precursor of the OV-6 positive hepatocytes that arose within the nodule. In the continuous diethylnitrosamine regimen a different staining pattern was seen. T-6 positive hepatocytes first appeared in periportal areas by the 5th week. These cells increased in numbers during the later weeks and with rare exceptions neither acquired the OV-6 epitope nor completely lost the H-4 epitope. Most HCC derived by the continuous diethylnitrosamine regimen were T-6 positive and OV-6 negative, suggesting a direct lineage from the periportal T-6 positive hepatocytes. These findings indicate that the lineage and phenotype of chemically induced HCC may vary with the carcinogenic regimen used and that HCC which arise in nodules may originate from cell types other than typical nodular cells.

Canalicular and mitochondrial membranes were investigated as early foci of hepatocyte injury in fed and fasted male Sprague-Dawley rats given 50 mg of 1,1-dichloroethylene (DCE)/kg. Staining of the bile canaliculi localized enzymes, leucine aminopeptidase (LAP), and Mg++-dependent ATPase (Mg++-ATPase), was examined by histochemistry in frozen sections. Mitochondrial membrane enzymes, including succinate dehydrogenase, also were examined by histochemistry. Staining of two monoclonal antibodies, C-1 and 9-B1, whose binding is localized in the bile canalicular region, was examined by immunofluorescence in frozen sections. Fasted rats treated with DCE developed moderate liver damage by 4 hours as evidenced by increases in serum transaminase and bilirubin, whereas fed rats developed only slight cell damage. Centrolobular loss of immunocytochemical and histochemical canalicular staining, especially for C-1 and Mg++-ATPase, was evident as early as 1 hour after DCE and was striking by 2 hours in both fed and fasted rats. Decreases in mitochondrial enzymes were not evident histochemically in fed animals at any time after DCE and were found only at the later times in fasted animals given the toxin. Thus, DCE administration to fed rats provides a new model system of selective bile canaliculi injury.