Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: Study of immunological memory and long-term toxicology

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types <it>in vitro</it>. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed.</p> <p>Results</p> <p>The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.</p> <p>Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8⁺T cell memory responses, whereas MVA-encoded nef induced CD4⁺T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity.</p> <p>Conclusion</p> <p>This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4⁺or a CD8⁺T cell response depending on the choice of vector.</p

Concepts in DNA immunization overcoming viral diversity and enhancing plasmid immunogenicity

On April 23, 1984, the prominent scientist Robert Gallo held a historical press conference at the Department of Health and Human Services, Washington D.C., USA. He announced that his laboratory at the National Institutes of Health had over the last months isolated a retrovirus named Human T-cell Leukemia Virus type III (HTLV-III). The virus came from 48 patients in the homosexual community in San Francisco. The city had just been hit by the mysterious epidemic of acquired immunodeficiency syndrome (AIDS). HTLV-111 was later renamed human immunodeficiency virus (HIV). At the same meeting, Gallo further explained that his laboratory was able to grow large quantities of the virus in cell cultures and as a consequence it was stated that "we believe that the new process will enable us to develop a vaccine to prevent AIDS in the future ... we hope to have such a vaccine ready for testing in approximately two years." This thesis is printed on the very same day, twenty years later and the now mature field of HIV/AIDS vaccine development has still not discovered what exactly mediates protection against HIV infection, while we are still far away from a clinically useful vaccine. Why is this? What makes HIV so special when other virus diseases, like polio, can be recognized and eliminated by the immune system, and where vaccination works very well? The focal points of this thesis are two major problems in modem vaccine development. Many viruses exist in multiple subtypes or serotypes, a phenomenon that has serious implications for the choice of vaccine antigen. It is especially critical when trying to vaccinate against HIV, the surface structure (gpl20) of which presents immense antigenic variability. Moreover, modem genetic vaccines based on smaller units of the virus or consisting of multiple genes (combination genetic vaccine) are weak immunogens, this is the second problem that this thesis explores. More specifically, we have shown that removal of inhibitory elements in a DNA immunogen is of importance for efficient induction of immunity. Further, antibody responses to a DNA immunogen can be substantially enhanced if the genetic immunogen is coupled to a carrier, in our case the polyomavirus VP1 capsid. In combination with generally immunoactivating agents, for instance recombinant granulocyte macrophage colony stimulating factor (GM-CSF), the HIV envelope genes from multiple subtypes (A, B and C) can change the envelope DNA immunogen into a potent entity that induces high titers of broadly reactive antibodies as well as cellular responses. We also show that immunization with proteins followed by DNA immunogens, a strategy tentatively called "reverse prime-boost immunization" induces strong immunity. These findings will be further validated in human clinical trials within the near future. Last but not least, we have developed an HIV murine challenge model based on pseudotyped viral particles; combining the HIV genome and the murine leukemia virus (HIV/MuLV) envelope. This model resembles human acute primary HIV infection. Protection in this model can be ascribed to cellular immunity, in the complete absence of antibodies. Using this model, we have shown that primeboost immunization induces better protection against subtype homologous HIV challenge, than against heterologous exposure. The immunization strategies covered in this thesis describe the biological problems that face vaccine development in general and HIV vaccinology in particular. The problems and concepts illustrate why the statement by several scientists in the 1980s has proven to be somewhat premature

Killing Kinetics of Simian Immunodeficiency Virus-Specific CD8+ T Cells: Implications for HIV Vaccine Strategies

Both the magnitude and function of vaccine-induced HIV-specific CD8 + CTLs are likely to be important in the outcome of infection. We hypothesized that rapid cytolysis by CTLs may facilitate control of viral challenge. Release kinetics of the cytolytic effector molecules granzyme B and perforin, as well as the expression of the degranulation marker CD107a and IFN-γ were simultaneously studied in SIV Gag164-172 KP9-specific CD8+ T cells from Mane-A*10+ pigtail macaques. Macaques were vaccinated with either prime-boost poxvirus vector vaccines or live-attenuated SIV vaccines. Prime-boost vaccination induced Gag-specific CTLs capable of only slow (after 3 h) production of IFN-γ and with limited (50% of tetramer-positive CD8+ T cells) degranulation and granzyme B release. The cytolytic phenotype following live-attenuated SIV vaccinations were similar to that associated with the partial resolution of viremia following SIVmac251 challenge of prime-boost-vaccinated macaques, albeit with less IFN-γ expression. High proportions of KP9-specific T cells expressed the costimulatory molecule CD28 when they exhibited a rapid cytolytic phenotype. The delayed cytolytic phenotype exhibited by standard vector-based vaccine-induced CTLs may limit the ability of T cell-based HIV vaccines to rapidly control acute infection following a pathogenic lentiviral exposure

Vaccine-Induced T Cells Control Reversion of AIDS Virus Immune Escape Mutants

Many current-generation human immunodeficiency virus (HIV) vaccines induce specific T cells to control acute viremia, but their utility following infection with escape mutant virus is unclear. We studied reversion to wild type of an escape mutant simian-HIV in major histocompatibility complex-matched vaccinated pigtail macaques. High levels of vaccine-induced CD8(+) T cells strongly correlated with maintenance of escape mutant virus during acute infection. Interestingly, in animals with lower CD8(+) T-cell levels, transient reversion to wild-type virus resulted in better postacute control of viremia. Killing of wild-type virus facilitated by transient reversion outweighs the benefit of a larger CD8(+) T-cell response that only maintains the less fit escape mutant virus. These findings have important implications for the further development of T-cell-based HIV vaccines where exposure to escape mutant viruses is common

NK Cell Function and Antibodies Mediating ADCC in HIV-1-Infected Viremic and Controller Patients

Natural killer (NK) cells have been suggested to play a protective role in HIV disease progression. One potent effector mechanism of NK cells is antibody-dependent cellular cytotoxicity (ADCC) mediated by antiviral antibodies binding to the Fc gamma RIIIa receptor (CD16) on NK cells. We investigated NK cell-mediated ADCC function and the presence of ADCC antibodies in plasma from 20 HIV-1-infected patients and 10 healthy donors. The HIV-positive patients were divided into two groups: six who controlled viremia for at least 8 y without treatment (controllers), and 14 who were persistently viremic and not currently on treatment. Plasma from both patient groups induced NK cell IFN-gamma expression and degranulation in response to HIV-1 envelope (Env) gp140-protein-coated cells. Patient antibodies mediating ADCC were largely directed towards the Env V3 loop, as identified by a gp140 protein lacking the V3 loop. Interestingly, in two controllers ADCC-mediating antibodies were more broadly directed to other parts of Env. A high viral load in patients correlated with decreased ADCC-mediated cytolysis of gp140-protein-coated target cells. NK cells from both infected patients and healthy donors degranulated efficiently in the presence of antibody-coated HIV-1-infected Jurkat cells. In conclusion, the character of ADCC-mediating antibodies differed in some controllers compared to viremic patients. NK cell ADCC activity is not compromised in HIV-infected patients.Funding Agencies|Swedish Research Council||Swedish International Development Cooperation Agency (SIDA)||Swedish Physicians Against AIDS Research Foundation|