Biotecnologia Aplicada
Elfos Scientiae
ISSN: 0684-4551
2000
Biotecnología Aplicada 2000;17:251-257

Biotecnología Aplicada 2000;17:251-257

XIII Conferencia Internacional sobre SIDA

Carlos Duarte

Centro de Ingeniería Genética y Biotecnología. AP 6162. CP 10600, La Habana. Cuba.

Code Number: ba00075

 Introducción

La XIII Conferencia Internacional sobre SIDA se celebró entre el 9 y el 14 de julio del 2000 en la ciudad sudafricana de Durban. Indudablemente, se crearon las mayores expectativas por ser este evento científico el primero de su tipo que se celebra en un país del Tercer Mundo. En particular, la República Sudafricana se ubica en el área geográfica más duramente golpeada por esta epidemia y 19,9% de su población adulta está infectada por el virus de la inmunodeficiencia humana (VIH).

En la actualidad, existen 34 millones de personas infectadas en el mundo y cada día trae un saldo de 16 000 víctimas nuevas. En la ausencia de un tratamiento capaz de curar la enfermedad, por los elevados costos de las terapias actuales y sus importantes efectos adversos, se reconoce ampliamente que la aplicación de una vacuna eficaz y de bajo costo es la única posibilidad de proteger del sida a la mayor parte de la población pobre e indefensa del planeta.

No es casual entonces que los organizadores hicieran énfasis, dentro del programa científico del evento, en el desarrollo de vacunas preventivas contra el VIH. En especial, se abordaron, con una profundidad no vista anteriormente, los aspectos de organización necesarios para el desarrollo y la evaluación de candidatos vacunales en el mundo, especialmente para África. También se subrayó la importancia de buscar nuevas fórmulas que estimulen las investigaciones en este campo y la participación de las grandes compañías farmacéuticas en estos trabajos.

Panorama actual de la evaluación clínica de candidatos vacunales. Aspectos estratégicos, financieros y de organización

La Tabla resume la situación actual de los principales candidatos.

Tabla. Los 16 principales candidatos vacunales en evaluación.

No.

Compañía

Candidato

Fase

País

Subtipo

Comentarios

1

VaxGen

Gp120

III en curso

USA/TAI

B y E

No se espera protección importante

2

Aventis Pasteur

Canarypox

II terminada

USA

B

Fase II con resultados no muy buenos

Fase I

Uganda

B

Muy baja respuesta CTL

Fase I-II
para 2000

Brasil, Haiti
y Trinidad

B

Con variante de virus mejorado con un gen
de VV que aumenta la expresión

3

Aventis Pasteur

Gp140 trimérica subtipo B

Fase I

USA/TAI

B

Su superioridad teórica sobre gp120 monomérica no está clara en la práctica

4

Biovector Therap. SA-Bioquest Inc

Lipopéptidos- CTL

Fase I

Francia

B

Debe pasar a fase II

5

Merck

DNA gag

Fase I

USA

B

No se conocen los resultados

6

Wyatt Lederle

DNA env-rev

Fase I

USA

B

Muy segura pero con baja inmunogenicidad

7

Cell-Sci

HPG30 (p17)

Fase I

USA

B

Es un péptido sintético p17. Malos resultados

8

IHV (Institute for Human Virology)

Toxoide Tat

Fase I

USA

B

Segura e inmunogénica, debe pasar a fase II

9

Chiron

DNA-alphavirus replicon

Preclínica

USA/África

B/C

Financiada con 24 000 000 USD por NIH

10

Univ Roma

DNA- Tat

Monos

Italia

B

Muy buena protección. Debe pasar a fase I

11

Oxford

DNA/MVA (subtipo A)

Monos

Kenya

A

IAVI financiará fase I en Kenya e Inglaterra

12

Australia

DNA/FPV (Fowlpox Virus)

Monos

Australia

B

Fase I en 2001 financiada por NIH
con 16 000 000 USD

13

Alphavax Corp

VEE (Encefalitis Equina Venezolana)

Monos

Sudáfrica

C

IAVI - fase I en Sudáfrica en 2001

14

IHV

Salmonella . vector DNA

Monos

Uganda

A

IAVI - fase I próxima en Uganda y Baltimore

15

Targeted Genetics

AAV (Virus Adeno Asociado)

Monos

África

A y C

IAVI finaciará fase I en África

16

NIH/Walter Reed

DNA/MVA

Monos

USA/TAI

E

Fase I inicia este año en EE. UU. solo con MVA

Es preciso destacar el papel activo que en este aspecto ha desempeñado la Iniciativa Internacional de Vacuna de SIDA (IAVI, del inglés International AIDS Vaccine Iniciative). Según su director Seth Berkely, la IAVI ha logrado reunir alrededor de doscientos millones de dólares estadounidenses (USD) y ha establecido un programa para la evaluación acelerada de candidatos vacunales para África. Su principal papel ha sido conectar centros de investigación o compañías biotecnológicas en países industrializados con países de África, y financiar proyectos conjuntos que van desde las etapas de investigación y desarrollo hasta la evaluación clínica de los candidatos vacunales. La IAVI se propone acelerar lo más posible el programa de desarrollo y evaluación de nuevos candidatos vacunales contra el sida. Para esto ha propuesto, entre otras medidas:

- comprimir las Fases I y II en un solo estudio;
- conducir en paralelo diferentes estudios de eficacia en diferentes países;
- evaluar hasta 25 candidatos vacunales y priorizar entre ellos a través de estudios en paralelo para llegar con 6 u 8 a Fase III antes del 2007.
- invertir entre 900 000 000 y 1 200 000 000 de USD extra para maximizar la posibilidad de éxito.

Como se refleja en la Tabla, la IAVI está financiando activamente cuatro candidatos vacunales que serán evaluados en África (Tabla, No. 11, 13-15). En un futuro cercano, planean incorporar entre 6 y 8 candidatos nuevos a su esquema de evaluación acelerada.

La labor desplegada por la IAVI se suma con fuerza a la desarrollada en la misma cuerda por la organización de las Naciones Unidas para el SIDA (ONUSIDA). La ONUSIDA comenzó un trabajo hace varios años para crear las condiciones para la realización de estudios clínicos de Fase III en Uganda, Tailandia y Brasil.

Situación de África

Como se conoce, la situación de África es la más trágica del mundo. Sin embargo, hasta hace un año no se había comenzado a evaluar ningún candidato vacunal para este continente. Pero la situación actual ya es diferente. El primer estudio clínico de Fase I se efectuó en Uganda con la vacuna de Aventis Pasteur (Tabla, No. 2), aunque ésta, en realidad, no incluye los subtipos de virus que circulan en Uganda y por eso ha sido muy criticada. Los resultados de este ensayo fueron presentados en una sesión de "late breakers" y se observó un nivel muy bajo de respuesta citotóxica contra el VIH en los vacunados.

Veamos ahora la situación por países.

República Sudafricana

En este país circulan cepas del subtipo C de forma mayoritaria. Aún no se han reportado muchas cepas recombinantes. Es el país con mejor infraestructura de investigación en África. El Departamento de Salud ha creado el proyecto SAVI (South African Vaccine Iniciative), que rige los esfuerzos en esa dirección.

Las investigaciones de vacunas se desarrollan fundamentalmente en la Universidad de Ciudad del Cabo y el Instituto Nacional de Virología. En estos momentos trabajan en cuatro enfoques:

- vacunas de ADN;
-  virus Vaccinia cepa Ankara modificada (MVA);
- BCG recombinante;
- "partículas semejantes a virus" producidas en plantas y hongos.

Paralelamente, comenzará en el 2001 la evaluación de un candidato vacunal basado en la tecnología generada por la compañía Alphavax (Tabla, No. 13, y reporte "Alphavirus Vaccine Vectors for Lentiviruses Targeted to Dendritic Cells In Vivo"). Se trata de un virus de la encefalitis equina (VEE) recombinante para los genes del VIH de una cepa del subtipo C aislada en Sudáfrica (reporte "Design and Development of a VEE Replicon Vector HIV-1-subtype C Vaccine for South Africa").

 Uganda

Circulan cepas recombinantes de forma mayoritaria, principalmente AG. Fue uno de los países en que la ONUSIDA trabajó inicialmente para crear las condiciones necesarias para evaluar vacunas y el único país africano en que se ha evaluado un candidato vacunal (reporte "HIV-1 Vaccine Trial in Uganda: Experience on Pre-and Post-vaccination Screening of Volunteers"). En el futuro se evaluará una vacuna oral de Salmonella (Tabla, No. 14).

Kenya

Recombinantes; AG fundamentalmente. Al igual que en la República Sudafricana el trabajo de vacunas es estructurado por un comité denominado KAVI, del inglés Kenyan AIDS Vaccine Initiative. Preparan un ensayo clínico de MVA (Tabla, No. 11) en colaboración con Oxford. Este ensayo incluye el gen gag y un gen multiepitópico de CTL.

 América

Sigue siendo Brasil, junto con Trinidad y Tobago y Haití, los sitios de elección para la realización de estudios clínicos por su incidencia y por la infraestructura creada. Las cepas predominantes son B al igual que en Cuba. En breve se debe iniciar un estudio clínico Fase I-II en estos tres países, con una nueva variante de Canarypox desarrollada por Aventis Pasteur (Tabla, No. 2).

Asia

Hasta el momento, Tailandia marcha a la vanguardia en la organización de los ensayos clínicos. Allí circulan los subtipos B y BE recombinantes. Ya se encuentra en marcha la Fase III de la vacuna de VaxGen con muy buena organización. La fase de reclutamiento está prácticamente terminada, al igual que el ensayo paralelo de esta vacuna que se lleva a cabo en los Estados Unidos (reporte "Successful Recruitment and Conduct of the First HIV Vaccine Efficacy Trial in North America and Europe"). Otros países como la India, tienen la potencialidad para iniciar estos estudios en el futuro.

Oceanía

Australia se prepara para comenzar la evaluación clínica de un candidato vacunal que es una combinación de ADN con poxirus (primer boost). Ésta será una de las primeras pruebas de este novedoso concepto, que también se ha aplicado al MVA y a otros virus. La preparación australiana, que ha resultado inmunogénica en ratones y macacos, coexpresa los genes gag y pol del VIH con el de IFN gamma (reporte "DNA and Fowlpoxvirus [FPV] HIV Vaccines Co-expression of Interferon-gamma by FPV Further Boosts HIV Specific T Cell Responses").

Nuevos enfoques vacunales y hallazgos básicos en torno a vacunas

Si bien el Congreso fue fructífero en los aspectos más prácticos de la instrumentación de ensayos clínicos, no se destacó por la abundancia de nuevos enfoques vacunales o reportes básicos de interés para el campo. Se pueden destacar dos reportes que demuestran la presencia de actividad CTL para virus de diferentes subtipos (reportes "Investigation of Cross-clade Cellular Immune Responses in Ugandan Patients Infected with Non-clade B HIV-1 by the ELISPOT Assay" y "Cross-clade HIV Specific CTL Study and CTL Epitope Mapping in Clade E/A HIV Infected Thais"). Estos hallazgos nuevos fortalecen la hipótesis en boga que postula que la respuesta CTL desempeña un papel protagónico en la protección contra el VIH.

Un enfoque vacunal original fue presentado por J. Lisziewicz (reporte "Novel HIV Vaccine for Induction of T Cell Immunity") al transfectar células dendríticas (DC del inglés dendritic cells) con complejos entre un plásmido que expresa proteínas del VIH y polietilenimina-manosa, e inmunizar macacos con estas DC autólogas para obtener una respuesta CTL fuerte y duradera de tipo Th1. Más interesante aún fue que la aplicación tópica de estos complejos en ratones depilados fue capaz de inducir respuesta CTL en los animales.

Los mejores resultados de vacunas con ADN en primates son los reportados por Barbara Ensoli en Roma. Se inmunizaron macacos con plásmidos que expresan el gen tat y quedaron protegidos contra el reto con el virus SHIV. La respuesta CTL, un patrón de linfocinas Th1 y el factor antiviral de células CD8 (CAF, del inglés cellular antiviral factor) correlacionaron con la protección.

Una aumento impresionante de la inmunogenicidad de la gp120 se encontró con el uso del adyuvante QS21, lo cual permite reducir notablemente las dosis de proteína. Sin embargo, se encontraron efectos adversos graves en los voluntarios que no permiten recomendar este compuesto aún para su uso en humanos (reporte "Low Dose Immunization Using the Adjuvant QS21 Combination with Recombinant MN and/or Bivalent MN/A244 [E Type] gp120 in HIV-1 Uninfected, Low-risk Volunteers [AVEG 036]").

Por último, se deben destacar algunas variantes novedosas de vacunas vivas con el empleo del virus adeno asociado (reporte "Adeno-associated Virus Vector-Based Vaccine Induces Long-term Humoral and Cell-mediated Immunity against Human immunodeficiency Virus") y el virus de la rabia (reporte "Recombinant Rabies Virus as Live Viral Vaccines for HIV-1"), con la inducción de respuesta tanto humoral como CTL en ratones.

 

Selected abstracts from the XIII International Conference on AIDS.
July 9–14, 2000, Durban, South Africa.

 

Alphavirus Vaccine Vectors for Lentiviruses Targeted to Dendritic Cells In Vivo

Johnston R,1 MacDonald G,1 Richmond E,1 Aronson J,2 Johnson P,3 Davis N1

1University of North Carolina, Chapel Hill, NC 27599 7290, United States.
Tel.: (1 919) 966 35 07; Fax: (1 919) 962 81 03; E-mail: rjohnst@med.unc.edu
2University of Texas Medical Branch, Galveston, TX, United States.
3Children’s Hospital Research Foundation, Columbus, OH, United States.

The efficacy of a vaccine vector system is influenced by the anatomical site and particular cell type in which expression occurs. Vaccine vectors based on the alphavirus Venezuelan equine encephalitis virus (VEE) have been employed successfully in a number of animal models of disease, including simian immunodeficiency virus [1, 2]. The VEE replicon RNA contains the gene of an immunogen in place of the structural protein genes. The replicon RNA is packaged into VEE replicon particles (VRP) by supplying the capsid and glycoprotein genes on helper RNAs in trans. VRP expressing GFP (gfp-VRP) and packaged in wild-type glycoproteins targeted efficiently to dendritic/Langerhans cells expressing DEC205 and MHCII surface markers. These cells were found in the subcapsular region of the draining lymph node within 1 h after subcutaneous inoculation. Single amino acid substitutions in the E2 glycoprotein dramatically altered these targeting characteristics. VRP packaged in the V3010 coat was inefficient in transit to the draining lymph node and was in small, round cells in the medulla of the node. Targeting to dendritic cells was restored in a second site revertant of V3010 (V3533), although V3533 targeted a broader range of dendritic cell types than wild-type. For reasons of safety, the VRP used in the successful VEE vector vaccine experiments to date were packaged in the glycoproteins of V3014, a VEE mutant carrying two attenuating point mutations. While low doses (103 IU) of V3014-packaged VRP targeted to lymph nodes, expression was in small, round cells in the medulla. At the higher doses used in the vaccination experiments (105–107 IU), targeting to dendritic cells was observed. The efficiency of the VRP in inducing an immune response was enhanced when the immunizing VRP were packaged in glycoproteins that targeted them to dendritic cells in vivo.

1. Pushko, et al. J Virol 1997;239:389–401.

2. Davis, et al. J Virol 1999;74:371–8.

 

Design and Development of a VEE Replicon Vector HIV-1-subtype C Vaccine for South Africa

Olmsted R,1 Karim SA,2 Williamson C,3 Morris L,4 Swanstrom R,5 Fiscus S,5 Frelinger J,5 Johnston R5

1AIphaVax, Inc. 770 W. Main Street, Durham, NC 27701, United States.
Tel.: (1 919) 688 6902; Fax: (1 919) 688 7396; E-mail: olmsted@alphavax.com 
 2CERSA, Durban, South Africa
3University of Cape Town, Cape Town, South Africa.
4National Institute of Virology, Johannesburg, South Africa.
5University of North Carolina, Chapel Hill, NC, United States.

Background. An international collaborative effort to design and develop an HIV-1-subtype C vaccine for use in South Africa was initiated in October 1998 under the support of the International AIDS Vaccine Initiative.

Methods. The candidate vaccine will be comprised of a mixture of Venezuelan equine encephalitis virus (VEE) replicon particles individually expressing HIV-1 gag, pol or env genes. The three genes were selected based on homology to the consensus sequence generated from primary isolates obtained from recent seroconvertors in Kwazulu/Natal.

Results. Sequence analyses identified the selected genes as subtype C, reflecting the predominate subtype that exists in potential clinical trial sites. Virus replicon particle (VRP) preparations expressing each of the three genes were prepared for in vitro and in vivo characterization. Western blot analysis of Gag-VRP infected cell lysates demonstrated high-level expression of authentic p55. Importantly, two subcutaneous inoculations of 105 infectious units of Gag-VRP induced both vigorous antigen specific CTL and humoral responses in Balb/c mice. Similarly, Pol-VRP and Env-VRP-infected cells expressed protein products of predicted size and immunoreactivity. Immunogenicity studies in mice have been initiated with Pol- and Env-VRP and results will be presented.

Conclusion. The individual components of the candidate VEE replicon HIV-1-subtype C vaccine have been engineered and characterized. Our goal is to begin Phase I clinical evaluation in the United States and South Africa within the next year.

 

HIV-1 Vaccine Trial in Uganda: Experience on Pre- and Post-vaccination Screening of Volunteers

Serwanga J,1 Kaleebu P,2 Mugerwa R,3 Mugyenyi P,4 Mbidde E,5 Andersson K,4 Flores J,6 Ellner J,7 Hom DL8

1Uganda Virus Research Institute. PO BOX 49, Entebbe, Uganda.
Tel.: + 256 41 320 385; Fax: + 256 41 321 137; E-mail: arbovir@infocom.co.ug 
2Uganda Virus Research Institute, Entebbe, Uganda.
3Makerere University, Kampala, Uganda.
4Joint Clinical Research Center, Kampala, Uganda.
5Uganda Cancer Research Institute, Kampala, Uganda.
6NAIDS/DAIDS/CDB, Rockville, United States.
7University of Medicine and Denistry, Newark, NJ, United States
8Case Western Reserve University, Cleveland, OH, United States.

Background. Uganda initiated a phase I placebo- controlled, double-blind, randomized HIV-1 preventive vaccine trial in early 1999. Forty healthy low-risk volunteers were randomized to receive recombinant ALVAC vCP205 HIV vaccine (n = 20), or 1 of 2 placebos. We present issues related to pre- and post-vaccination screening.

Objectives. 1) To establish criteria for pre-screening of volunteers; 2) to design an algorithm to differentiate vaccine-related immune responses from natural infection.

Methods. Prior to recruitment, screening was done using two EIAs, Cambridge EIA and Welcozyme EIA and a Western blot (WB) (Cambridge Biotech). Those with indeterminate WB, a second test was carried out on earlier stored (3 months) sera, in addition p24 gag or pol DNA PCR was performed. To differentiate vaccinees from natural HIV infection an algorithm that initially uses peptide based ELISAs that lack a portion of gp41 present in the vaccine construct i.e. the Sanofi (Genetic Systems Corp) and Select-HIV (Biochem Corp) was used. If positive or discordant, WB, DNA PCR (p24 gag) and viral load (Roche, Amplicor 1.5) were performed to confirm infection. In addition, viral typing by HMA or sequencing, and viral culture on a second sample were performed.

Results. At screening all 40 volunteers were HIV negative. One individual became naturally infected during the trial. This subject was positive on both the Sanofi and Select EIA kits, was DNA PCR positive, and had a plasma viral load 18,000 copies/mL, with viral culture and viral typing underway to confirm acute HIV infection.

Conclusion. HIV diagnostic tests must be carefully selected for volunteer screening and differentiating vaccine-related immune responses from natural infection. The algorithm we instituted is adequate but labour intensive and expensive. Alternative simple algorithms need to be developed and validated before large efficacy trials.

 

Successful Recruitment and Conduct of the First HIV Vaccine Efficacy Trial in North America and Europe

Harro C,1 Judson F,2 Brown SJ,3 Marmor M,4 Li E,5 Alonzo G,5 Gulati V,5 Berman PW,5 Francis D5

 1Johns Hopkins University, Center for Immunization Research, Hampton House, Room 117, 624 N. Broadway Baltimore, MD, United States. Tel.: +1 410 614 49 37; Fax: +1 410 955 27 91; E-mail: charro@jhsph.edu 
2Department of Public Health, Denver, CO, United States.
3AIDS ReSEARCH Alliance, West Hollywood, CA, United States.
4New York University, New York, NY, United States.
5VaxGen, lnc, Brisbane, CA, United States.

Introduction. Since the early 1980s, the HIV epidemic has severely impacted men who have sex with men (MSM). Vaccine preparedness studies in MSM demonstrated seroincidence (> = 1.5%/year), willingness to participate, and follow-up rates that are supportive of efficacy trials.

Methods. This is a randomized, double blind, placebo controlled, multi-center trial. Volunteers were screened for high-risk sexual activity, confirmed HIV-1 seronegative by HIV-1 ELISA, and randomized in 2:1 ratio to AIDSVAX(r) B/B (300  mg each antigen) or placebo at month 0, 1, 6, 12, 18, 24, and 30. Baseline data include medical history, demographics, risk assessment, and motivations for participation. Follow-up safety, immunogenicity, behavioral, and social harm data are collected and monitored by the Data and Safety Monitoring Board.

Results. A total of 5415 volunteers (94% male; mean age 36) were enrolled from June 1998 to October 1999 at 61 sites in North America and Europe. A total of 311 high-risk women were included for safety and immunogenicity analysis. Volunteers are mostly white (83%) and well educated (61% with college level education or higher). Altruism is the major motivation for trial participation; 99% want to help find an effective HIV vaccine, and 75% want to obtain HIV research information. Baseline MSM sexual behavior indicates 54% with 5 or more male partners and 44% with HIV+ partners in the past 6 months. Through January 2000, follow-up is excellent with > 98% retention. Immunizations are well tolerated with no serious adverse events attributed to vaccine.

Conclusions. Evaluation of HIV vaccine efficacy requires the strong support and cooperation of a high-risk population. Enrollment of this trial demonstrates that sufficient numbers of high-risk MSM and women are willing to participate, are altruistically motivated, and exhibit high levels of compliance to date. An interim efficacy analysis is anticipated in 2001.

 

DNA and Fowlpoxvirus (FPV) HIV Vaccines. Co-expression of Interferon-gamma by FPV Further Boosts HIV Specific T Cell Responses

Kent S,1 Zhaol A,1 Dale CJ,1 Land S,2 Boyle D,3 Ramshaw I4

 1Macfarlane Burnet Centre, Yarra Bend Rd, Fairfield 3078, Australia.
Tel.: +61 392 822 175; Fax: +61 394 826 152; E-mail: kentbur@net.edu.au 
2National Serology Reference Laboratory, Fitzroy, Australia. 
3CSIRO Animal Health, Geelong, Australia.
4Australian National University, Canberra, Australia.

Background. A preventive HIV vaccine would be a quantum advance on current efforts to control the HIV pandemic. While HIV protein vaccines have not shown encouraging efficacy and live attenuated vaccines are unsafe, both plasmid DNA and fowlpoxvirus (FPV) HIV vaccines can induce CTL responses in a proportion of outbred primates, although neither is particularly efficacious alone.

Methods. A DNA/FPV prime/boost HIV-1 vaccine regimen has been studied in macaques and a rFPV expressing both HIV-1 gag/pol and human interferon- g (FPVgag/pol-IFN g) was constructed and assessed for safety and ability to enhance HIV-primed T cells in macaques (Macaca nemestrina).

Results. CTL and Th1 responses were dramatically enhanced in both mice and monkeys following DNA/FPV prime/boost regimens. High level CTL and Th responses to both Env and Gag proteins were observed in all studied monkeys following immunisation. Inoculation of DNA/FPV vaccinated animals with 100 monkey infectious doses of HIV-1 resulted in very rapid clearance of HIV-1, almost certainly mediated by HIV-specific CTL/Th1 responses. Recent studies have demonstrated that novel FPV vaccines co-expressing IFN g could be even more effective in boosting HIV-primed T cells. FPV gag/pol-IFN g vaccinations were safe and generated enhanced T cell proliferative responses (of a Th1 phenotype) to HIV antigens but not control tetanus antigens. Enhanced CTL responses to HIV antigens were also observed following FPVgag/pol-IFNg expressing vaccinations.

Conclusions. These observations suggest that priming with DNA vaccines and boosting with avipox co-expressing IFN g should induce unprecendented levels HIV-specific T cells and could be useful therapeutic or preventive HIV-1 vaccines.

An urgent investment in moving these regimens through the clinical trials process could reap significant dividends to humanity.

 

Investigation of Cross-clade Cellular Immune Responses in Ugandan Patients Infected with Non-clade B HIV-1 by the ELISPOT Assay

Rutébemberwa A,1 Auma B,2 Gillmour J,3 Jones G,3 Watera C,4 Imami N,3 Kaleebu P,4 Whitworth J,5 Gotchs F6

1Uganda Virus Research Institute. PO Box 49, Entebbe, Uganda.
Tel.: +256-41-320272/320042; Fax: +256-41-321137; E-mail: mrc@starcom.co.ug 
2Medical Research Council (MRC)/UVRI, Entebbe, Uganda.
3Chelsea and Westminster Hospital, London, United Kingdom.
4MRC/UVRI, Entebbe, Uganda.
5MRC, Entebbe, Uganda.
6Chelsea and Westminster Hospital, London, Uganda.

Background. An effective HIV vaccine should induce good cross-clade cellular immune responses. In order to prepare for future vaccine trials, there is need to investigate the extent of cross-clade immune responses in HIV-1 infected persons, and to develop simple and rapid assays to study cellular immunity.

Methods. A cohort of 50 HIV-1-infected persons was established in Entebbe, Uganda. Infecting clades were determined by env HMA, and gag sequencing is underway. All persons were HLA tissue-typed by SSP-PCR. Longitudinal and cross-clade cellular immune responses have been investigated by peptide and vaccinia Elispot assays.

Results. The MHC class I molecules common in the cohort are HLA A (38%), A29 (14%), A30 (38%), B58 (33%), B45(17%), and B57 (17%). In 21 persons in whom viral subtypes were determined by env HMA, 11 were clade A, 9 were clade D and 1 was clade C. The following peptides were found to induce the highest T-cell responses: A2 restricted p17 gag peptides SLYNTVATL derived from clade B (LAI) and D, and SLFNTVATL derived from clade A; B57 restricted gag peptides ISPRTLNAW derived from LAI/D, LSPRTLNAW derived from clade A and TSTLQEQIGW derived from LAI/D. Most patients recognised peptide constructs derived from subtypes other than their own subtype. Consistent hierarchies of recognition were observed in peptide responses in 4/6 persons. 50% of 27 persons (5 not tissue typed) responded to vaccinia expressing gag, pol or env constructs of HIV-1 MN, 111B, A or D.

Conclusion. We have shown that cross-clade cellular immune responses exist in HIV-1 infected persons, however, dominant peptide recognition is apparent.

Elispot assays are simple, rapid assays that can be used in large studies of HLA typed subjects, in addition vaccinia Elispot assays can be used successfully in non-tissue typed subjects to measure global responses to HIV antigens expressed by rVV.

 

Recombinant Rabies Virus as Live Viral Vaccines for HIV-1

Pomerantz R , Foley H, Schnell M, Siler C, Dietzschold B, McGeytigan J

Thomas Jefferson University, 1020 Locust St., Suite 329 JAH, Philadelphia, PA 19107, United States.
Tel.: +1 215 503. 85 11 75; Fax: +1 215 923 19 56; E-mail: roger.j.pomerantz@mail.tju.edu

To develop a live-virus strategy in the development of HIV-1 vaccines recombinant replication-competent rabies virus (RV) vaccine strain-based vectors expressing HIV-1 envelope protein (gp160) were constructed from both a laboratory-adapted (CXCR-4-tropic) and a primary (dual-tropic) HIV-1 isolate. An additional transcription stop/start unit within the RV genome was used to express HIV-1 gp160 in addition to other RV proteins. The HIV-1 gp160 protein was stable and functionally expressed, as indicated by Western blotting and fusion of human T-cell lines after infection with the recombinant RV expressing the HIV-1 envelope protein. Inoculation of mice with the recombinant RVs expressing HIV-1 gp160 induced a strong humoral response directed at the HIV-1 envelope protein after a single boost with recombinant HIV-1 gp120 protein. Moreover, high neutralization titers up to 1 to 800 against HIV-1 could be detected in the serum of mice primed with RV-expressing HIV-1 envelope proteins and boosted once with recombinant HIV-1 gp120. Preliminary data also indicate cross neutralization titers against other strains of HIV-1 by the same sera and high levels of specific cytotoxic T-lymphocytes (CTLs). These recombinant RVs express HIV-1 glycoproteins on the infected cells surfaces and may expose unique fusion-based epitopes. This approach has several unique characteristics which may make it an efficacious vector for a vaccine. These include low-level replication in a variety of human cell-types, altered non-pathogenic properties due to deletion of the rabies glycoprotein, lack of integration of a viral vaccine genome, and potential unique viral apitope exposure to the immune system. Of note, this report represents the first development of a non-segmented RNA virus as a HIV -1 vaccine vector. The present data indicate that a live recombinant RV, a rhabdovirus, expressing HIV-1 gp160 may serve as.

 

Adeno-associated Virus Vector-Based Vaccine Induces Long-term Humoral and Cell-mediated Immunity against Human Immunodeficiency Virus

KE-QIN Xin,1 Masashi Urabe,2 Jun Yang,3 Kenji Hamajima,3 John Mor Katsuji Okuda,5 Keiya Ozawa,2 Kenji Okuda3

13-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Tel.: 81-45-787-2602; Fax: 81-45-786-2509; E-mail: kqxin@med.yokohama-cu.ac.jp 
2Tochigi, Japan.
3Yokohama, Japan.
4Avigen, Inc., Alameda, CA, United States.
5Department of Microbiology, Tokyo Dental College, Chiba, Japan.

Background. Adeno-associated virus (AAV) vector has been widely used for gene therapy in a variety of preclinical studies. The virus has infected most of the population and never shown any pathogenic effects in humans. AAV vector which can integrate into the cellular DNA has been shown to confer long-term gene expression in a number of tissues. In this study, the immunogenicity of the vector containing HIV envelope gene (AAV HIVenv) was examined in animal models.

Methods. The AAV vectors expressing HIV env (AAV-HIVenv) and murine IL-2 (AAV-IL2) were constructed by the calcium phosphate co-precipitation method. AAV-HIVenv vector (106–1011 particles) with or without AAV-IL2 vector (1010 particles) was intramuscularly injected once into BALB/c mice. AAV-HIVenv vector (5x108–5x1013 particles) was intramuscularly injected into Japanese macaque monkeys on day 0 and 30.

Results. A single injection of 1010 particles of AAV-HIVenv vector induced strong HIV-1-specific serum IgG (log214), fecal IgA (log212) antibodies and cell mediated immunity (CMI) including cytotoxic T lymphocyte activity (25% specific cell lysis at E/T ratio of 80) and delayed-type hypersensitivity (DTH) response (13.6x10-2 mm) in BALB/c mice. These titers have been stable for ten months. Moreover, co-administration of AAV-HIVenv vector with AAV-IL2 vector significantly enhanced HIV-1-specific CMI. Boosting with AAV-HIVenv vector in the 10th month after immunization strongly enhanced the humoral and CMI responses. Furthermore, the mouse antisera could strongly inhibit the replication of HIV-1 subtype B strains (32%–58%). The injection of AAV-HIVenv vector into Japanese macaque monkeys also induced high levels of humoral immunity and fairly high levels of CTL responses.

Conclusions. These results demonstrate that AAV vector is useful for HIV vaccine development.

 

Novel HIV Vaccine for Induction of T Cell Immunity

Lisziewicz J

Research Institute for Genetic and Human Therapy at Georgetown University,
Med-Den SW 307, 3900 Reservoir Road, NW, Washington DC, 20007, United States.
Tel.: +1 202 687 2833; Fax: +1 202 687 2987; E-mail: right@gunet.georgetown.edu

Background. HIV-specific cell-mediated immune responses (T helper and CTL) correlate with protection. Antigen-presenting dendritic cells (DC) are the most efficient cells to induce potent cell-mediated immune responses.

Methods. We used genetically modified dendritic cells (GM-DC) to provide efficientt antigen presentation for naive T-cells. GM-DC were generated from from monocyte-derived DC transduced with a plasmid DNA encoding a replication and integration defective HIV vector using a non-viral gene delivery system (polyethylenimine-mannose). Immunogenicity of GM-DC was tested in vitro and in vivo in non-human primates.

Results. After a single stimulation, GM-DC elicited in vitro a potent activation of autologous naive CD4 and CD8 T-cells (up to 8% of CD3 gated cells expressed intracellular IFN-g) and generated vigorous CTL responses (27% of p55-specific lysis, E:T = 100:1). Restimulation of primed T cells with HIV-antigen presenting cells confirmed the antigen-specificity of these immune responses. Autologous GM-DC injected into pigtailed macaques induced prompt, vigorous, and longlasting CTL responses against HIV. Three weeks after a single immunization, animals presented a vigorous effector CTL response (up to 50% of gag-specific lysis in the absence of in vitro antigenic restimulation, E:T = 100:1). Low activity of effector CTL were still found 7 months later (up to 12% of gag-specific, lysis E:T = 100:1). At this time, vigorous memory CTL response was detected (up to 40% of gag-specific lysis after antigenic stimulation, E:T = 40:1). Interestingly, no antibodies were detected, suggesting that GM-DC raised a pure Th1 type of immune response. No toxic side effects were documented.

Conclusions. We provide evidences that a novel genetic immunization with GM-DC can induce unprecedented vigorous and long lasting CTL.

 

Low Dose Immunization Using the Adjuvant QS21 Combination with Recombinant MN and/or Bivalent MN/A244 (E Type) gp120
in HIV-1 Uninfected, Low-risk Volunteers (AVEG 036)

Evans T,1 McElrath MJ,2 Gorse G,3 Mulligan M,4 Grahams B,5 Schwartz D,6 Montefiori D,7 Francis D,8 Flores D9

1University of Rochester, 601 Elmwood Avenue, Box 689, Rochester, NY, 14642, United States.
2University of Washington, Seattle, WA, United States.
3University of St. Louis, St. Louis, MO, United States.
4University of Alabama, Birmingham, AL, United States.
5Vanderbilt University, Nashville, TN, United States.
6Johns Hopkins University, Baltimore, MD, United States.
7Duke University, Durham, NC, United States.
8VaxGen Corporation, Brisbane, CA, United States.
9DAIDS, NIH, Bethesda, MD, United States.

Background. The saponin adjuvant QS21 has a dose-sparing effect in HIV-1 envelope immunization in HIV-uninfected human volunteers. However, the lowest immunogenic dose of gp120 used in combination with QS21 was not determined, and local reactogenicity was remarkable.

Methods. A study was undertaken in 60 HIV-1 seronegative, low-risk volunteers using a QS21 (100 mg) formulation buffered with polysorbate 80 in combination with recombinant HIV-1 MN gp120, 3 mg or 0.5 mg (VaxGen), 6 mg bivalent MNgp120/A244 (Thai E), or placebo, administered on a 0, 1, 6 month schedule. Assessment of local pain, tenderness, and systemic signs or symptoms were performed after each immunization, and binding and neutralizing antibodies were measured two weeks after immunization.

Results. The number of complaints of severe local pain and tenderness following any vaccination fell from 37% (7/19) in a previous low-dose QS21 study to 10% (6/60) in this study. Binding Ab responses were measured to MN rgp 120 and MN V3 loop were similar to those achieved historically with 300  mg of MN gp120. HIV-1 MN neutralization GMTs following the 2nd and 3rd immunizations were: 0.5 mg group (QS21 adjuvant) - 23 and 723, 3 mg (QS21 adjuvant) - 94 and 359, 3 mg (QS21 plus 6 mg alum) - 206 and 383, bivalent MN/A244 (QS21 - plus 12 mg alum) - 103 and 241. Placebo plus QS21 adjuvant - < 10. Of note the highest 2 individual post third immunization titers (> 3,000) achieved were in the lowest dose group (0.5 mg). No Env-specific CTL activity was detected using a chromium release assay.

 

Cross-clade HIV Specific CTL Study and CTL Epitope Mapping in Clade E/A HIV Infected Thais

Buranapraditkun S,1 Ruxrungtham K,2 Warachit P,3 Honda M,4 Phanuphak P2

1Chulalongkorn University, Faculty of Medicine, Department of Medicine,
Chulalongkorn University, Bangkok 10330, Thailand.
Tel.: + 66-2-254-2323; Fax: + 66-2-254-7577; E-mail: bsuprane@chula.ac.th
2Chulalongkorn University, Bangkok 10330, Thailand.
3Thai NIN, Nonthaburi, Thailand.
4National Institute of Infectious Diseases, Tokyo, Japan.

Background. There are 2 clades of HIV-1: E/A and B (B’ > B) found in Thailand. Cross-clade CTL study and epitope mapping are warranted for HIV vaccine development to be tested in Thailand.

Objectives. To find the prevalence of cross-clade CTL activities in HIV-infected Thais with CD4+ > 300 cell/ mL. And to identify common and/or novel HIV-1 CTL gag epitopes.

Methods. Classic Cr51 release CTL assays were performed in 22 HIV-1-infected Thai patients. Recombinant HIV-1 gag and pol vaccinia for both clade A and B were used. HIV-1 clade A gag truncated peptides (aa 1–499, consensus sequence of 92UG037, each is 20 aa in length, with 10-aa overlaps between sequential peptides) were used for gag-CTL epitope mapping. The plasma samples were serotyped for HIV-1 subtypes.

Results. Mean CD4+ cell counts 528 cells/ mL, median plasma HIV-RNA 6510 copies/mL (range < 50–52, 935). >From serotyping, 10 patients were infected with clade E, the rest awaits for further genotyping. All the patients showed CTL activity against clade A gag and pol (10/10, 100%), whereas 80% and 30% revealed cross-reactive CTL against clade B gag and pol, respectively. There are 6 epitopes identified in 3 evaluated patients. Four epitopes locate in p24 region and 2 in p17 region. Based on the Los Alamos HIV immunology database, 4 of them are restricted by HLA B8, A2, and A26. However, there are 2 epitopes that requires further HLA restriction study.

Discussions. In HIV vaccine design for CTL induction, the breadth of cross clade reactivity needs to be considered. Our results indicate that HIV clade A-gag provided much higher CTL cross-reactivity than HIV clade A-pol. Thus, HIV-1 gag clade A is suitable to be included in a candidate HIV vaccine to be evaluated in Thailand.

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