Perspectivas de curación: DermaVir, una vacuna de ADN con efecto terapéutico contra el VIH/sida y desarrollada racionalmente / Towards a cure: DermaVir, a rationally designed therapeutic DNA vaccine against HIV/AIDS

Hallazgos recientes en el área de la intensificación de los tratamientos antirretrovirales demuestran que la forma de llegar a una curación para el VIH exige la inclusión de nuevas estrategias de tratamiento como las vacunas terapéuticas. La vacuna terapéutica DermaVir incluye elementos tecnológicos clave en el diseño de una vacuna racional: un único inmunógeno plásmido de ADN (pADN) que expresa a 15 antígenos del VIH, formulación de nanomedicina y una única administración tópica de la vacuna enfocada a las células dendríticas. Luego de su administración tópica sobre la piel preparada, las células epidérmicas de Langerhans activadas transportan la nanomedicina DermaVir a los nódulos linfáticos para expresar a los antígenos del VIH codificados como pADN e inducir a las células T precursoras/de memoria con una alta capacidad de proliferación. Se han demostrdo la seguridad, la inmunogenicidad y la eficacia preliminar de la DermaVir en varios modelos animales y en humanos con infección por VIH. Esta novedosa tecnología de vacunación terapéutica podría constituir un nuevo paradigma en el trtamiento contra el VIH.

Recent findings on the field of antiretroviral treatment intesnsification demonstrate thar the way towards a cure for HIV requires the involvement of novel treatment strategies like therapeutic vaccines. DermaVir therapeutic vaccine includes key technological elements of rational vaccine design: a single plasmid DNA (pDNA) immunogen that expresses 15 HIV antigens, nanomedicine formulation and a unique dendritic cell-targeting topical vaccine administration. Following topical administration on the prepared skin, DermaVir nanomedicine is transported by activated epidermal Langerhans cells to the lymph nodes to express the pDNA-encoded HIV antigens and induce precursor/memory T cells with high proliferation capacity. Safety, immunogenicity and preliminary efficacy of DermaVir have been demonstrated in several animal models and HIV-infected human subjects. This novel therapeutic vaccination technology might offer a new treatment paradigm against HIV.

HIV-1 binding and neutralizing antibodies of injecting drug users

Previous studies have demonstrated a stronger seroreactivity against some synthetic peptides responsible for inducing neutralizing antibodies in injecting drug users (IDU) compared to that of individuals sexually infected with HIV-1 (S), but the effectiveness in terms of the neutralizing ability of these antibodies has not been evaluated. Our objective was to study the humoral immune response of IDU by determining the specificity of their antibodies and the presence of neutralizing antibodies. The neutralization capacity against the HIV-1 isolate MN (genotype B), the primary HIV-1 isolate 95BRRJ021 (genotype F), and the seroreactivity with peptides known to induce neutralizing antibodies, from the V2 and V3 loops of different HIV-1 subtypes, were analyzed. Seroreactivity indicates that IDU plasma are more likely to recognize a broader range of peptides than S plasma, with significantly higher titers, especially of V3 peptides. Similar neutralization frequencies of the MN isolate were observed in plasma of the IDU (16/47) and S (20/60) groups in the 1:10 dilution. The neutralization of the 95BRRJ021 isolate was more frequently observed for plasma from the S group (15/23) than from the IDU group (15/47, P = 0.0108). No correlation between neutralization and seroreactivity with the peptides tested was observed. These results suggest that an important factor responsible for the extensive and broad humoral immune response observed in IDU is their infection route. There was very little difference in neutralizing antibody response between the IDU and S groups despite their differences in seroreactivity and health status.

Human immunodeficiency virus type 1 neutralization by plasma from B or F genotype infected individuals

Anti-human immunodeficiency virus type 1 (HIV-1) "binding antibodies" (antibodies capable of binding to synthetic peptides or proteins) occur throughout HIV-1 infection, are high-titered and highly cross-reactive, as confirmed in this study by analyzing plasma from B and F genotype HIV-1 infected individuals. Plasma from individuals infected with clade F HIV-1 displayed the most frequent cross-reactivity, in high titers, while Bbr plasma showed much higher specificity. Similarly, neutralization of a reference HIV-1 isolate (HIV-1 MN) was more frequently observed by plasma from F than B genotype infected individuals. No significant difference was seen in neutralization susceptibility of primary B, Bbr or F clade HIV-1 by plasma from individuals infected with the classical B (GPGR) or F HIV-1, but Bbr (GWGR) plasma were less likely to neutralize the F genotype primary HIV-1 isolates. The data indicate that both B and F genotype derived vaccines would be equally effective against B and F HIV-1 infection, with a slightly more probable effectiveness for F than B genotype. Although the Bbr variant appears to induce a much more specific humoral immune response, the susceptibility in neutralizing the Brazilian HIV-1 B genotype Bbr variant is similar to that observed with the classical B genotype HIV-1.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins / Reconocimiento por anticuerpos de péptidos sintéticos que imitan regiones inmunodominantes de las proteínas p24 y p17 de VIH-1

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant a-helical structure and perform important functions throughout thevirallife-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration.In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic a-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzymeimmunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short a-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C- terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accesibility to the minimal epitopes byspecific antibodies, in solution.

El gen gag del VIH-1 codifica una región de 55kDA que contiene tres subdominios: matriz (p17), cápside (p24) y nucleocápside (p15). Las proteínas p24 y p17 tienen una estructura predominante helicoidal y cumplen un rol importante en el ciclo de vida del virus. En este trabajo presentamos los resultados de inmunorreactividad de péptidos sintéticos que imitan regiones helicoidales de p24 y p17. Utilizando enzimoinmunoensayos se evaluó la influencia de modificaciones en las secuencias nativas sobre la capacidad de reconocimiento de anticuerpos específicos en solución y en fase sólida, incluyendo el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes mínimos. La conformación de los péptidos se determinó por dicroísmo circular y los resultados se correlacionaron con los de inmunorreactividad. Se observó que la capacidad de reconocimiento de anticuerpos por péptidos pequeños que imitan estructuras helicoidales de p24 y p17 mejoró con el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes. Estas modificaciones mejoran la inmovilización sobre las superficies sólidas y permiten una mayor accesibilidad de los anticuerpos a los epitopes mínimos en solución.

Cytotoxic T lymphocyte responses to vaccinia virus antigens but not HIV-1 subtype E envelope protein seen in HIV-1 seronegative Thais.

The HIV-1 prime boost phase I/II vaccine trial using a recombinant canarypox vector, vCP1521, containing subtype E env (gp120), and subtype B env (gp41), gag and protease has started in Thailand. We have demonstrated that although 4 from 15 human immunodeficiency virus type 1 (HIV-1) seronegative Individuals showed cytotoxic T lymphocyte (CTL) responses to vaccinia virus antigens, none of them showed specific CTL responses to subtype E Env after in vitro stimulation. This preliminary study suggests that specific CTL responses to subtype E envelope detected in HIV-1 seronegative Individuals after vaccination should be considered as specific responses to the immunization.

Anticuerpos neutralizantes frente a 5 cepas de VIH-1 en macacos inmunizados con el polipéptido multiepitópico TAB9 / Neutralizing antibodies to 5 HIV-1 strains in macaques immunized with the multi-epitope polypeptide TAB9

Presence of neutralizing antibodies(Nab) to 5 laboratory strains of human immunodeficiency virus type-1 (HIV-1) was studied and compared in the sera of eight macaca (Macaca fascicularis) after the third and fourth immunizations with multi-epitope polypeptide TAB9, emulsified with adjuvant Montanide ISA 720. Four animals were inoculated 1 mg and the others 200 micrograms, 2 animals were used as controls and injected only with adjuvant. Although the presence of neutralizing antibodies to homologous strains of HIV-group group B was confirmed in most animals after immunization through antigen p24 capture ELISA (DAVIH-Agp24, Cuba), no statistically significant differences were found neither in titers caused by antigen concentrations nor in the responses after the third and fourth immunizing doses. Controls did not develop neutralizing antibodies.

Further evaluation of alternative strategy for HIV testing in India.

A total 320 sera from groups at risk for HIV were evaluated by two (198 specimens) or three (122 specimens) screening tests for confirmatory anti-HIV testing in comparison to Western Blot as gold standard. Sera positive by both screening tests showed 100% correlation with Western Blot although with a false positivity rate of 3.2%. In specimens positive by 1st screening test but negative by the second, (considered negative for anti-HIV antibody as per WHO algorithm), 8.7% were found to be Western Blot positive showing the serious problem of false negativity of the proposed WHO algorithm. Employing the system of three screening test systems did not provide additional benefit for the specimens positive by initial two screening tests since all of them were positive by third test also. However, the study involving three screening tests substantiated the need for Western Blot in discordant specimens (i.e. positive by first test but negative by second), since in this group one out of 22 (4.6%) such specimens were Western Blot positive. Considering the serious consequences of both false positive and false negative results, it is felt that alternative strategy of confirmatory anti-HIV serology, although economical may not be suitable substitute for Western Blot in India at this juncture when the prevalence of HIV infection is relatively low.

Respuesta inmune humoral hacia VIH-2 en México / Humoral immune response to HIV-2 in Mexico

Objetivo. Estudiar las reacciones hacia antígenos de VIH-2 de sueros mexicanos VIH-1 positivos, en relación con la vía de transmisión y el estado clínico de los individuos infectados. Material y métodos. Se estudiaron 654 sueros (492 VIH-1 positivos y como controles 162 VIH-1 negativos). Se preparon Inmuno blots (IB) con antígenos semipurificados de MS-VIH-2 y de IIIb/LAV-VIH-1 y se corrieron en ambos todas las muestras de suero. Resultados. En el análisis de los IB de los sueros VIH-1 positivios se encontró que 79 por ciento (388/492) presentaron reactividad con, por lo menos, una proteína VIH-2; 71 por ciento (352/492) de los sueros reconocen la proteína de cápside p24; la glicoproteína transmembranal de VIH-2 es reconocida por 24 por ciento (119/492) de los sueros y 9 por ciento (44/492) reconocieron la glicoproteína externa de este retrovirus. La reactividad con VIH-2 de sueros VIH-1 positivos está significativamente asociada a la vía de adquisición de la infección (81 por ciento en vía sexual contra 39 por ciento en vía sanguínea) y al estado clínico (84 por ciento en pacientes asintomáticos o con linfadenopatía contra 31 por ciento en pacientes con SIDA). Se descartó la posible infección con los dos tipos de VIH por medio del ensayo comercial Liatek (Organon Teknica). Conclusiones. Se propone como hipótesis que las cepas introducidas originalmente en nuestro país a través de las dos vías principales de transmisión son de orígenes diferentes

Objective. To study the reactions of Mexican HIV-1 positive sera to HIV-2 antigens, and their relation to the mode of ransmission and the clinical status of infected individuals. Material and Methods. Six-hundred and fifty-four sera samples collected in Mexico were tested using HIV-2 immunoblot (IB) techniques; 492 samples were from HIV-1 positive cases and 162 from HIV-1 negative controls. Results. Seventy-nine percent (388/492) of the HIV-1 reactive sera showed reactivity with at least one HIV-2 protein: 71% (352/492) recognized the capsid protein p24, 24% (119/492) the transmembrane glycoprotein and 9% (44/492) the external glycoprotein of HIV-2. Considering the transmission mechanism, HIV-2 reactivity occurred in 81% (324/401) of the sexually infected patients, and only in 39% (16/41) of people infected through blood products. Ten highly reactive gp32 HIV-2 sera samples were titrated and results showed that reactivity with HIV-1 gp41 was always higher than that to HIV-2 gp32. HIV-1-HIV-2 dual infection was discarded by negative results with the commercial assay Liatek HIV 1+2. Conclusions. We propose that the serological cross-reactivity found can be due to a possible initial introduction in Mexico of two different viral strains through the two main ransmission routes and that the strains circulating in sexually infected individuals are more similar to the HIV-2 strains, at least with respect to their glycoproteins, than the HIV-1 strains predominant in other countries.