Receptor Binding by Cholera Toxin B-Subunit and Amino Acid Modification Improves Minimal Peptide Immunogenicity.

We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75-84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes).

Increased expression and immunogenicity of HIV-1 protease following inactivation of the enzymatic activity.

HIV-1 protease is an important target for anti-HIV therapy but has not received much attention as a vaccine antigen. To investigate the immunogenic properties of HIV-1 protease, we designed DNA plasmids encoding variants of the protease gene. Mutations resulting in enzymatic inactivation (D25N) and resistance to standard antiretroviral drugs (V82F/I84V) were introduced in order to examine the impact of the enzymatic activity on immunogenicity and the possibility to induce immune responses against drug resistant protease, respectively. The enzymatic inactivation of protease resulted in significantly increased in vitro expression as well as in vivo immunogenicity. The inactivated protease was highly immunogenic in both BALB/c and HLA-A0201 transgenic C57Bl/6 mice, and the immunogenicity was retained when the gene was delivered as a part of a multigene HIV-1 DNA vaccine. The drug resistance mutations hampered both the cellular and humoral immune responses, as the mutations also affect both CD4 and CD8 T cell epitopes. Taken together, our data demonstrates the possibility to drastically increase the immunogenicity of HIV-1 protease.

Potent cross-reactive immune response against the wild-type and drug-resistant forms of HIV reverse transcriptase after the chimeric gene immunization.

HIV reverse transcriptase (RT) can be considered as a target and an instrument of immunotherapy aimed at limiting the emergence and spread of drug-resistant HIV. The chimeric genes coding for the wild-type and multi-drug-resistant RT (RT1.14) fused to lysosome-associated membrane protein 1 (LAMP-1) were injected intramuscularly into BALB/c mice. The immune response was assessed by ELISpot, cytokine ELISA intracellular IFN-gamma staining, and antibody ELISA. The genes for RT- and RT1.14-LAMP fusions (RT-LAMP and RT1.14-LAMP) were immunogenic generating a mixed Th1/Th2-profile of immune response, while the wild-type RT gene induced only weak immune response. Specific secretion of Th1-cytokines increased with increasing level of RT modification: RT

HIV-1 reverse transcriptase artificially targeted for proteasomal degradation induces a mixed Th1/Th2-type immune response.

Targeting of a DNA vaccine encoded protein for degradation via the proteasome is attempted since it may enhance the immunogenicity of the vaccine. We have fused HIV-1 reverse transcriptase (RT) to mouse ornithine decarboxylase (ODC), a protein rapidly degraded by proteasome in an ubiquitine-independent fashion, to enhance the introduction of RT into the MHC class I pathway. We also designed a fusion of RT with two short signals from the C-terminus of ODC (ODCsig) representing a minimal proteasome-targeting moiety of ODC (PEST signal). Fusion to ODC or ODC signal domain led to a marked enhancement of RT degradation. Plasmids encoding RT-ODC and RT-ODCsig chimera were used to immunize BALB/c mice. The administration of the plasmids was not associated with autoimmune disease. Moreover, mice receiving RT-ODCsig gene mounted a mixed Th1/Th2 response characterized by the in vitro secretion of IFN-gamma, IL-2, TNF-alpha, IL-4, and IL-10 upon stimulation of splenocytes with RT protein or RT derived peptides. Serum titers of 10(2) to 10(3) were observed in more than 50% of animals in that group, whereas fewer animals mounted an anti-RT response in the RT-ODC gene immunized group. Chimeras of the type described here can, therefore, be used in vaccinations aiming to induce HIV-1 RT-specific immune response.

Murine models for HIV vaccination and challenge.

HIV-1 only infects humans and chimpanzees. SIV or SHIV are, therefore, used as models for HIV in rhesus, cynomologus and pigtail macaques. Since conducting experiments in primate models does not fully mimic infection or vaccination against HIV-1 and is expensive, there is a great need for small-animal models in which it is possible to study HIV-1 infection, immunity and vaccine efficacy. This review summarizes the available murine models for studying HIV-1 infection with an emphasis on our experience of the HIV-1-infected-cell challenge as a model for evaluating candidate HIV-1 vaccines. In the cell-based challenge model, several important factors that, hopefully, can be related to vaccine efficacy in humans were discovered: the efficiency of combining plasmid DNA representing several of the viral genes originating from multiple clades of HIV-1, the importance of adjuvants activating innate and induced immunity and the enhanced HIV eradication by drug-conjugated antibody.

Vaccination against drug resistance in HIV infection.

HIV-1 resistance to currently employed antiretroviral drugs and drug-associated adverse reactions and toxicity point to a need for additional measures to control HIV-1 replication in HIV-infected patients. The immune system of HIV-infected individuals mount an immune response against the regions harboring drug-resistance mutations, sometimes stronger than that against the parental wild-type sequences. A potent cross-reactive immune response against drug-resistant pol proteins can suppress the replication of drug-escaping HIV. This suggests the possibility for a vaccination against existing and anticipated drug-resistant HIV variants. If successful, therapeutic vaccines against drug resistance would ease the therapeutic modalities and limit the spread of drug-resistant HIV. A better understanding of the complex interactions between patterns of drug-resistance mutations, immune responses against these mutations and their antigen presentation by particular human lymphocyte antigen alleles could help to tailor these vaccines after new drugs/new mutations. In this review, we describe the developments in the field of immunization against mutations conferring drug resistance and evaluate their prospects for human vaccination.

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

BACKGROUND: 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 in vitro. 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. RESULTS: The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.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. CONCLUSION: 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.

A new multi-clade DNA prime/recombinant MVA boost vaccine induces broad and high levels of HIV-1-specific CD8(+) T-cell and humoral responses in mice.

The results presented here are from the preclinical evaluation in BALB/c mice of a DNA prime/modified vaccinia virus Ankara (MVA) boost multi-gene multi-subtype human immunodeficiency virus-1 (HIV-1) vaccine intended for use in humans. The plasmid DNA vaccine was delivered intradermally using a Biojector, and the MVA was delivered intramuscularly by needle. This combination of recombinant DNA and MVA proved to induce extraordinarily strong cellular responses, with more than 80% of the CD8(+) T cells specific for HIV-1 antigens. Furthermore, we show that the DNA priming increases the number of T-cell epitopes recognized after the MVA boost. In the prime/boost-immunized animals, a significant proportion of CD8(+) T cells were stained positive for both interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), a feature that has been associated with control of HIV-1 infection in long-term non-progressors. The HIV-1-specific antibody levels were moderate after the plasmid DNA immunizations but increased dramatically after the MVA boost. Although the initial injection of MVA induced significant levels of vaccinia-neutralizing antibodies, the HIV-specific responses were still significantly boosted by the second MVA immunization. The results from this study demonstrate the potency of this combination of DNA plasmids and MVA construct to induce broad and high levels of immune responses against several HIV-1 proteins of different subtypes.

Immunization with HIV protease peptides linked to syngeneic erythrocytes.

New potent vaccine adjuvants are desirable for increasing the efficacy of novel vaccine modalities such as DNA and peptides. We therefore tested if syngeneic erythrocytes could serve as delivery vectors for selected HIV peptides and compared the potency of these constructs to immunization with peptides in phosphate buffered saline or in incomplete Freunds adjuvant. Immunization of mice with peptides in a low dose (5 ng) coupled to erythrocytes induced a weak immune response in mice. These peptides alone (5 microg) gave no immune responses, while formulating the peptides (50 microg) in IFA induced strong homologous immunity as well as prominent cross reactivity to a related mutant epitope. Thus, vaccine delivery using syngeneic erythrocytes, although attractive for clinical use, might be of limited value due to the low amount of antigen that can be loaded per erythrocyte.

Evaluation of immunogenicity and efficacy of combined DNA and adjuvanted protein vaccination in a human immunodeficiency virus type 1/murine leukemia virus pseudotype challenge model.

A DNA plasmid encoding human immunodeficiency virus type 1 (HIV-1) env, nef and tat genes was used in mice in a prime-boost immunization regimen with the corresponding recombinant proteins. The genetic immunogen was delivered with a gene gun and the proteins were injected intramuscularly together with the adjuvant AS02A. Immunizations were followed by experimental challenge with pseudotyped HIV-1 subtype A or B virus. In an initial experiment in which animals were challenged four weeks after the final immunization, all single modality and prime-boost vaccinations resulted in a significant level of protection as compared to control animals. There was a trend for DNA-alone immunization yielding the highest protection. In a subsequent study, a late challenge was performed 19 weeks after the final immunization. All groups having received the DNA vaccine, either alone or in combination with adjuvanted protein, exhibited strong protection against HIV replication. The subtype-specific protection against the experimental HIV challenge was significantly stronger than the cross-protection. Cellular and humoral immune responses were assessed during immunization and after challenge, but without clear correlation to protection against HIV replication. The data suggest that either DNA or protein antigens alone provide partial protection against an HIV-1/MuLV challenge and that DNA immunization is essential for achieving very high levels of efficacy in this murine HIV-1 challenge model. While prime-boost combinations were more immunogenic than DNA alone, they did not appear to provide any further enhancement over DNA vaccine mediated efficacy. The DNA immunogen might prime low levels of CD8+ T cells responsible for virus clearance or possibly a yet unidentified mechanism of protection.

Reduced cellular immune responses following immunization with a multi-gene HIV-1 vaccine.

We investigated the effects of immunizing with several genes and subtypes of HIV-1. The genes used as immunogens were: gp160 envelope (env subtypes A, B and C), p37gag (gag subtypes A and B), rev (subtype B) and reverse transcriptase (RT subtype B). The different genes are all carried by separate plasmids. C57BL/6 and BALB/c mice were immunized with different combinations of the genes together with recombinant cytokine granulocyte macrophage-colony stimulating factor. The env genes injected alone induced significantly stronger cellular responses to envelope in both strains of mice than when env genes were injected together with gag and RT genes. In the C57BL/6 mice, the envelope specific responses were significantly increased after spatial separation of env genes from gag and RT genes as compared to when all vaccine genes were injected as a mixture. The gag responses were strong in gag-immunized animals and were not significantly affected by the spatial separation of gag and RT genes from the env genes. Our results illustrate the importance of being cautious when formulating multivalent genetic vaccines and that it might be possible to overcome lost immune responses through spatial separation of vaccine antigens.

HIV-1 reverse transcriptase targeted for proteasomal degradation as a prototype vaccine against drug-resistant HIV-1.

Acquisition of drug-resistance conferring mutations leads to an enhanced degradation of HIV-1 reverse transcriptase (RT) affecting its immunogenicity. The mechanism of this degradation is not known. We investigated the input of proteasome in this degradation, and explored a possibility to enhance the proteasomal degradation of RTs to potentiate the immunogenic peformance of RT genes. To this end, a C-terminal fusion was made of RT with ornithine decarboxylase (ODC) that is rapidly degraded by proteasome in an ubiquitine-independent fashion. Eukaryotic cells were transiently transfected with the genes for wild-type (wt) RT, multi-drug-resistant (MDR) RT, and their chimeras with ODC. RT expression in the presence or absence of the proteasome inhibitors MG132 and epoxomicin was quantified by Western blotting. Treatment with MG132 led to a two-fold increase in the level of wtRT, and a four-fold increase in the level of MDR-RT accumulation. Treatment with epoxomicin had virtually no effect on the accumulation of wtRT, while stabilizing MDR-RT two-fold. Since epoxomicin is a more specific proteasome inhibitor, it indicated that degradation of wtRT may not be solely proteasomal. Fusion to ODC considerably decreased the intracellular levels of both RT-ODC and MDR-RT-ODC as compared to parental proteins. MG132 treatment increased the intracellular RT-ODC content 20-fold (up the level of the MG132-treated wtRT; 60-80 fg/cell), and epoxomicin treatment, 10-fold as compared to non-treated samples. Thus, attachment of ODC moiety has modified the metabolic pathway of RT targeting it to proteasomal degradation. We are currently testing if this is translated into an enhanced MHC class I performance of wild-type and drug-resistant RTs in gene immunization.

Immunological cross-reactivity against a drug mutated HIV-1 protease epitope after DNA multi-CTL epitope construct immunization.

Epitopes in HIV polymerase were analyzed by peptide binding to human leukocyte antigen (HLA) A0201 molecules, the most frequent HLA class in the Caucasian population. We found that HIV-1 protease peptides representing both the wild type and anticipated drug resistance variants of the sequence bound well to HLA-A0201. We also found that wild type as well as a double mutated variant of the epitope was strongly immunogenic in HLA-A0201 transgenic mice, either as individual peptides or encoded in DNA multi-CTL epitope constructs. Immunological cross-reactivity between different variants of the peptide could be seen, suggesting that it may be possible to induce a broad immune response by immunizing with drug resistance-mutated epitopes. This may be of advantage for HIV-1 infected patients since such a response may cause a better outcome of an anti-retroviral drug therapy.

Multigene/multisubtype HIV-1 vaccine induces potent cellular and humoral immune responses by needle-free intradermal delivery.

Gene vaccination encounters problems different from those of gene therapy since both a short half-life of the gene and a strong immune response to the gene product are desirable. We have evaluated a DNA vaccine consisting of seven plasmids encoding nine HIV-1 proteins. Using a needle-free delivery device, the Biojector, together with recombinant mouse GM-CSF, this vaccine induced strong gp160 Env- and p24 Gag-specific cellular and humoral immune responses in mice. The rGM-CSF was crucial for inducing both antibodies and antigen-specific CD8(+) T cell responses against both gp160 and p24. A GMP-produced lot of this vaccine, intended for human use, was delivered intradermally or intramuscularly into BALB/c mice at a GLP-accredited animal facility. This vaccine induced strong cellular responses independent of the route of immunization; moreover, no signs of toxicity were detected after histopathological examination of various tissues. Overall, the results indicate that the intradermal delivery of multigene/multisubtype HIV DNA in combination with recombinant GM-CSF is a safe and efficacious strategy for inducing high levels of specific CD8(+) T cells and unusually high titers of antibodies. This vaccine has been approved by the Swedish Medicinal Products Agency and is currently in a Phase I clinical trial.

Reverse transcriptase-based DNA vaccines against drug-resistant HIV-1 tested in a mouse model.

Drug resistance is becoming a problem in the treatment of the human immunodeficiency virus type one (HIV-1). To obtain therapeutic DNA vaccines that would target multiple drug-resistance (DR) mutations, we cloned genes for DR HIV-1 reverse transcriptase (RT) and codon-optimized synthetic genes encoding clusters of human CTL epitopes located at the sites of DR-mutations (RT minigenes) and antibody and CTL-epitope tags. Expression of RT genes/minigenes in eukaryotic cells was confirmed by Western blotting and immunofluoresence staining with RT- or tag-specific antibodies. Immunization of mice with DR-RT gene induced no RT-specific antibodies. Immunization of HLA-A(*)0201-transgenic mice with RT minigenes induced RT-specific cellular responses detected by interferon-gamma secretion. This documents first steps in creating therapeutic vaccine against drug-resistant HIV strains.