Anne Kjerrström

Multi-subtype gp160 DNA immunization induces broadly neutralizing anti-HIV antibodies

A highly desirable feature for an human immunodeficiency virus type 1 (HIV-1) vaccine is the ability to induce broadly reactive anti-envelope antibodies that can neutralize primary HIV-1 isolates. Two immunizations with an HIV-1 envelope-encoding plasmid together with recombinant granulocyte–macrophage colony-stimulating factor (rGM-CSF) resulted in high antibody titers in mice. The antibody induction was further enhanced after immunization with genes encoding HIV-1 envelopes originating from subtypes A, B and C. The sera from these animals were able to neutralize A, B and C viral isolates, whereas the sera from animals immunized solely with subtype B DNA neutralized only subtype B virus. The combined DNA vaccine gave serum antibodies with broad recognition of HIV-1 envelope epitopes as determined by peptide mapping. Cell-mediated immunity was not compromised by the increased humoral immunity. This demonstrates the ability of multiple envelope genes to induce the desired antibody response against several subtypes. Moreover, it documents the ability of rGM-CSF to enhance the potency of such a vaccine when given simultaneously. The strategy may be useful for making an HIV vaccine more potent and broadly effective against strains of different clades.

Gene Combination Raises Broad Human Immunodeficiency Virus-Specific Cytotoxicity

DNA plasmid immunization has the important advantage over traditional vaccines of making it possible to combine selected genes into one vaccine. The efficacy of a combination of DNA plasmids encoding the nef, rev, and tat HIV-1 regulatory genes in inducing cellular immune responses was analyzed in asymptomatic HIV-1-infected patients. Patients initially selected for having low or no detectable immune responses to Nef, Rev, or Tat antigens developed MHC class I-restricted cytolytic activities as well as enhanced bystander effects. The induction of memory cells against target cells infected with the whole HIV-1 genome was analyzed by using a pseudovirus HIV-1/murine leukemia virus (MuLV), and target cells infected with vaccinia virus carrying the respective gene. The most remarkable change observed after immunization with the gene combination was an increase in cytotoxic T lymphocyte (CTL) precursors to target cells infected with the whole HIV-1 genome. Infection by the pseudotype HIV-1/MuLV virus should result in a multitude of HIV-1 peptides presented on the target cell surface, representative of the in vivo situation. An in vitro assessment of the expression of the single and combined gene products showed that this was consistent with the induction of CTL responses in vivo. No clinical advantage or adverse effects were noted. Therapeutic effects of such immunization may become measurable by structured therapy interruption.

DNA-Encoding Enzymatically Active HIV-1 Reverse Transcriptase, but Not the Inactive Mutant, Confers Resistance to Experimental HIV-1 Challenge

The present study was undertaken to examine the immunogenicity of a single plasmid DNA representing the reverse transcriptase (RT) of HIV-1. Plasmids containing the enzymatically active RT as well as a mutated nonenzymatically active RT with nucleotide (nt)-binding motifs of YMDD and YMLL, respectively, were used to immunize mice. Both constructs induced similar good antibody and T cell responses, with a tendency towards antibody directed to peptides representing the active and mutated sites. Immunized mice were challenged with a murine pseudotype HIV-1/MuLV infected spleen cells. Seven out of 10 mice immunized with RT had no recoverable HIV-1, while 10 individuals immunized with the RT mutant and all the 18 controls had high levels of recoverable HIV-1. This indicates that mutation of RT reduces the desired immunogenicity.

DNA-plasmids of HIV-1 induce systemic and mucosal immune responses.

Abstract DNA-based immunization has been shown to induce protective immunity against several microbial pathogens including HIV-1. Several routes of DNA vaccination have been exploited. However, the properties of the immune responses seem to differ with the different routes used for DNA delivery, ultimately affecting the outcome of experimental challenge. We measured the primary immune response following one vaccination. This report presents differences associated with three different DNA delivery routes: intramuscular injection, intranasal application, and gene-gun based immunization. Induction of systemic humoral immune responses was achieved most efficiently by either intranasal or gene-gun mediated immunization, followed by intramuscular injection. Mucosal IgA was reproducibly induced by intranasal instillation of the DNA, and found in lung washings, faeces, and vaginal washings. Cytotoxic T cells were not induced by a single immunization, but were observed after three immunizations using intramuscular injections.

DNA vaccination in mice using HIV-1 nef, rev and tat genes in self-replicating pBN-vector.

The immunogenicity of a self-replicating DNA-vector containing HIV-1 nef gene (pBN-Nef) was characterized using various DNA delivery methods. In addition, gene gun immunisation was used for assessing immunogenicity of two other HIV-1 genes (rev and tat) given in the same vector. The pBN-Nef was the most immunogenic raising both humoral and cell-mediated immune responses in mice; these responses lasted for up to six months. The pBN-Nef vector was immunogenic also when given intramuscularly or intradermally. The pBN-Rev construct did not elicit humoral responses but did elicit proliferative as well as CTL-response against the corresponding protein. The pBN-Tat was a poor immunogen in all respects. The antibodies elicited with various DNA delivery methods belonged to different antibody subclasses; however, two main epitopes in Nef were frequently recognized by all of them.

Th1-biased immune responses induced by DNA-based immunizations are mediated via action on professional antigen-presenting cells to up-regulate IL-12 production

The efficacy of DNA‐based immunization in conferring protective immunity against certain microbial pathogens including human immunodeficiency virus type 1 (HIV‐1) has been described. The potential advantage of DNA‐based immunization over the traditional vaccines largely results from its capacity to efficiently induce Th1‐biased immune responses against an encoded antigen. We describe how Th1‐biased immune responses are induced by DNA‐based immunization, using a DNA vaccine construct encoding HIV‐1 gp160 cDNA and an eukaryotic expression plasmid carrying murine IFN‐γ cDNA. Transfection of an eukaryotic expression plasmid carrying immunostimulatory sequences (ISS) as well as a gene of interest (DNA vaccine) into professional antigen presenting cells (APC) induced transactivation of IL‐12 mRNA, which resulted in antigen‐specific Th1‐biased immune responses against the encoded antigen. Th1‐biased immune responses induced by DNA‐based immunization were substantially upregulated by a codelivery of an ectopic IFN‐γ expression system, and this augmentation was mediated via action on professional antigen presenting cells to upregulate IL‐12 production. Taken together, it appears likely that Th1‐biased immune responses induced by DNA‐based immunization are mediated via action on professional antigen‐presenting cells to produce IL‐12. Interestingly, the model provided strikingly resembles that previously described in infection with Listeria monocytogenes, an intracellular Gram‐positive bacterium that induces strong Th1‐biased immune responses. The result suggests that DNA‐based immunization mimics certain aspects of natural infection with microbial organisms like attenuated vaccines, which in turn provides a rationale to the question of why DNA‐based immunization so efficiently induces protective immunity against these microbial pathogens.