Effective Delivery of Nef-MPER-V3 Fusion Protein Using LDP12 Cell Penetrating Peptide for Development of Preventive/Therapeutic HIV-1 Vaccine.

BACKGROUND: There is no effective and safe preventive/therapeutics vaccine against HIV-1 worldwide. Different viral proteins such as Nef, and two regions of Env including; variable loop of gp120 (V3) and membrane proximal external region of gp41 (MPER) are particularly important for vaccine development in different strategies and they are also the primary targets of cellular and humoral immune responses. On the other side, LDP12 is a new cell-penetrating peptide (CPP) which is capable of therapeutic application and cargoes delivery across the cellular membrane. OBJECTIVE: In current study, we designed and produced Nef-MPER-V3 fusion protein harboring LDP12 that has the capability of being used in future vaccine studies. METHODS: The CPP-protein was expressed in E. coli Rosseta (DE3) strain and purified through Ni-NTA column. Characterization of cellular delivery and toxicity of the recombinant protein were evaluated by western blotting and MTT assay. RESULTS: Our results showed that the CPP-protein was successfully expressed and purified with high yield of 5 mg/L. Furthermore, non-cytotoxic effect was observed and specific band (~ 37 KDa) in western blotting indicated the capability of LDP12 to improve the rate of penetration into HEK-293T cells in comparison with a control sample. CONCLUSION: Altogether, the data indicated that LDP12 CPP could be utilized to internalize HIV-1 Nef-MPER-V3 protein into eukaryotic cell lines without any toxicity and represented a valuable potential vaccine candidate and this guarantees the further evaluation towards the assessment of its immunogenicity in mice, which is currently under process.

Consistent elicitation of cross-clade HIV-neutralizing responses achieved in guinea pigs after fusion peptide priming by repetitive envelope trimer boosting.

The vaccine elicitation of broadly neutralizing responses is a central goal of HIV research. Recently, we elicited cross-clade neutralizing responses against the N terminus of the fusion peptide (FP), a critical component of the HIV-entry machinery. While the consistency of the elicited cross-clade neutralizing responses was good in mice, it was poor in guinea pigs: after seven immunizations comprising either envelope (Env) trimer or FP coupled to a carrier, serum from only one of five animals could neutralize a majority of a cross-clade panel of 19 wild-type strains. Such a low response rate-only 20%-made increasing consistency an imperative. Here, we show that additional Env-trimer immunizations could boost broad FP-directed neutralizing responses in a majority of immunized animals. The first boost involved a heterologous Env trimer developed from the transmitted founder clade C strain of donor CH505, and the second boost involved a cocktail that combined the CH505 trimer with a trimer from the BG505 strain. After boosting, sera from three of five animals neutralized a majority of the 19-strain panel and serum from a fourth animal neutralized 8 strains. We demonstrate that cross-reactive serum neutralization targeted the FP by blocking neutralization with soluble fusion peptide. The FP competition revealed two categories of elicited responses: an autologous response to the BG505 strain of high potency (~10,000 ID50), which was not competed by soluble FP, and a heterologous response of lower potency, which was competed by soluble FP. While the autologous response could increase rapidly in response to Env-trimer boost, the heterologous neutralizing response increased more slowly. Overall, repetitive Env-trimer immunizations appeared to boost low titer FP-carrier primed responses to detectable levels, yielding cross-clade neutralization. The consistent trimer-boosted neutralizing responses described here add to accumulating evidence for the vaccine utility of the FP site of HIV vulnerability.

Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques.

The development of an effective AIDS vaccine has been challenging because of viral genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs). We engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to those of human bnAbs, and conferred complete immunity against a mixture of simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and they may be applicable for treatment of diverse diseases, including infections, cancer, and autoimmunity.

Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m, a novel potent CD4-antibody fusion protein against HIV-1.

We previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.

Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via intra- and paracellular pathways.

Facing the threat of highly variable virus infection, versatile vaccination systems are urgently needed. Intranasal mRNA vaccination provides a flexible and convenient approach. However, the nasal epithelium remains a major biological barrier to deliver antigens to nasal associated lymphoid tissue (NALT). To address this issue, a potent polymer-based intranasal mRNA vaccination system for HIV-1 treatment was synthesized using cationic cyclodextrin-polyethylenimine 2k conjugate (CP 2k) complexed with anionic mRNA encoding HIV gp120. The delivery vehicle containing CP 2k and mRNA overcame the epithelial barrier by reversibly opening the tight junctions, enhanced the paracellular delivery of mRNA and consequently minimized absorption of toxins in the nasal cavity. Together with the excellent intracellular delivery and prolonged nasal residence time, strong system and mucosal anti-HIV immune responses as well as cytokine productions were achieved with a balanced Th1/Th2/Th17 type. Our study provided the first proof of evidence that cationic polymers can be used as safe and potent intranasal mRNA vaccine carriers to overcome the nasal epithelial barrier. The safe and versatile polymeric delivery system represents a promising vaccination platform for infectious diseases.

In vivo study of targeted nanomedicine delivery into Langerhans cells by multiphoton laser scanning microscopy.

Epidermal Langerhans cells (LCs) function as professional antigen-presenting cells of the skin. We investigated the LC-targeting properties of a special mannose-moiety-coated pathogen-like synthetic nanomedicine DermaVir (DV), which is capable to express antigens to induce immune responses and kill HIV-infected cells. Our aim was to use multiphoton laser microscopy (MLM) in vivo in order to visualize the uptake of Alexa-labelled DV (AF546-DV) by LCs. Knock-in mice expressing enhanced green fluorescent protein (eGFP) under the control of the langerin gene (CD207) were used to visualize LCs. After 1 h, AF546-DV penetrated the epidermis and entered the eGFP-LCs. The AF546-DV signal was equally distributed inside the LCs. After 9 h, we observed AF546-DV signal accumulation that occurred mainly at the cell body. We demonstrated in live animals that LCs picked up and accumulated the nanoparticles in the cell body.

NK cell responses to simian immunodeficiency virus vaginal exposure in naive and vaccinated rhesus macaques.

NK cell responses to HIV/SIV infection have been well studied in acute and chronic infected patients/monkeys, but little is known about NK cells during viral transmission, particularly in mucosal tissues. In this article, we report a systematic study of NK cell responses to high-dose vaginal exposure to SIVmac251 in the rhesus macaque female reproductive tract (FRT). Small numbers of NK cells were recruited into the FRT mucosa following vaginal inoculation. The influx of mucosal NK cells preceded local virus replication and peaked at 1 wk and, thus, was in an appropriate time frame to control an expanding population of infected cells at the portal of entry. However, NK cells were greatly outnumbered by recruited target cells that fuel local virus expansion and were spatially dissociated from SIV RNA+ cells at the major site of expansion of infected founder populations in the transition zone and adjoining endocervix. The number of NK cells in the FRT mucosa decreased rapidly in the second week, while the number of SIV RNA+ cells in the FRT reached its peak. Mucosal NK cells produced IFN-γ and MIP-1α/CCL3 but lacked several markers of activation and cytotoxicity, and this was correlated with inoculum-induced upregulation of the inhibitory ligand HLA-E and downregulation of the activating receptor CD122/IL-2Rß. Examination of SIVΔnef-vaccinated monkeys suggested that recruitment of NK cells to the genital mucosa was not involved in vaccine-induced protection from vaginal challenge. In summary, our results suggest that NK cells play, at most, a limited role in defenses in the FRT against vaginal challenge.

Toxicology, biodistribution and shedding profile of a recombinant measles vaccine vector expressing HIV-1 antigens, in cynomolgus macaques.

As a new human immunodeficiency virus type 1 (HIV-1) vaccine approach, the live-attenuated measles virus (MV) Schwarz vaccine strain was genetically engineered to express the F4 antigen (MV1-F4). F4 is a fusion protein comprising HIV-1 antigens p17 and p24, reverse transcriptase and Nef. This study assessed the toxicity, biodistribution and shedding profiles of MV1-F4. Cynomolgus macaques were intramuscularly immunized one or three times with the highest dose of MV1-F4 intended for clinical use, the reference (Schwarz) measles vaccine or saline, and monitored clinically for 11 or 85 days. Toxicological parameters included local and systemic clinical signs, organ weights, haematology, clinical and gross pathology and histopathology. Both vaccines were well tolerated, with no morbidity, clinical signs or gross pathological findings observed. Mean spleen weights were increased after three doses of either vaccine, which corresponded with increased numbers and/or sizes of germinal centers. This was likely a result of the immune response to the vaccines. Either vaccine virus replicated preferentially in secondary lymphoid organs and to a lesser extent in epithelium-rich tissues (e.g., intestine, urinary bladder and trachea) and the liver. At the expected peak of viremia, viral RNA was detected in some biological fluid samples from few animals immunized with either vaccine, but none of these samples contained infectious virus. In conclusion, no shedding of infectious viral particles was identified in cynomolgus monkeys after injection of MV1-F4 or Schwarz measles vaccines. Furthermore, no toxic effect in relation to the MV vaccination was found with these vaccines in this study.

Robust antigen-specific humoral immune responses to sublingually delivered adenoviral vectors encoding HIV-1 Env: association with mucoadhesion and efficient penetration of the sublingual barrier.

The efficient induction of virus-specific mucosal antibodies is an important unmet objective in Human Immunodeficiency Virus Type-1 (HIV-1) vaccine research. One promising approach is sublingual (SL) immunization. We examined the effectiveness of SL delivery of two different viral vectors: (i) a recombinant adenovirus (rAd5), and (ii) a Herpes Simplex Virus Type-1 amplicon vector (HSV-1). Initial in vitro videomicroscopy experiments showed that rAd5 particles were trapped in saliva (i.e., that Ad5 was mucoadhesive) - unlike HSV-1 virions, which migrated freely in both saliva and water. In vivo imaging studies in mice revealed that only the rAd5 vector efficiently transduced the SL epithelium. Consistent with this, SL delivery of an rAd5 encoding HIV-1 envelope glycoprotein (Env) resulted in robust antigen-specific antibody responses in plasma and in vaginal washes, whereas SL delivery of a HSV-1 amplicon vector encoding HIV-1 Env failed to elicit Env-specific antibodies. In contrast, both vectors elicited equivalent humoral responses following intramuscular (IM) delivery. Finally, SL delivery of the rAd5:Env vector resulted in elevated levels of Env-specific serum IgA, and vaginal IgA and IgG, when compared to IM delivery of the same vector. These results findings shed light on vector properties (mucoadhesion, penetration of the sublingual barrier) which may be important for the induction of potent humoral immune responses following sublingual vector administration. Our data also show that SL delivery of an Env-encoding rAd5 vector can elicit a potent antigen-specific mucosal antibody response in the absence of adjuvant. Overall, these findings support the further exploration of the SL delivery route for HIV-1 vaccine delivery.

Immunogenicity, safety, biodistribution and persistence of ADVAX, a prophylactic DNA vaccine for HIV-1, delivered by in vivo electroporation.

ADVAX is a DNA-based candidate HIV vaccine that was safe but weakly immunogenic when delivered intramuscularly (IM) in humans. Studies were performed in animal models to determine whether an alternative delivery method, in vivo electroporation (EP), could improve the immunogenicity of ADVAX while maintaining an acceptable safety profile. Immunization of mice with ADVAX with or without EP at weeks 0, 3, and 6, revealed significantly higher gamma interferon ELISpot responses to all antigens in the EP groups. Antigen-specific CD4+ and CD8+ T cell responses, as quantified by intracellular cytokine staining, both improved significantly with EP. Evaluation of repeat-dose toxicity of ADVAX-EP in rabbits did not reveal any safety concerns. Biodistribution studies of ADVAX delivered IM and with EP in rats indicated that the vaccine was localized predominantly to the administration site in both groups. PCR-based quantitation of residual plasmid at Day 60 indicated that the potential for integration events into the host genome was low for both IM and EP delivery. Taken together, these data supported the clinical development of ADVAX delivered with EP in human volunteers.

Biodistribution, persistence and lack of integration of a multigene HIV vaccine delivered by needle-free intradermal injection and electroporation.

It is likely that gene-based vaccines will enter the human vaccine area soon. A few veterinary vaccines employing this concept have already been licensed, and a multitude of clinical trials against infectious diseases or different forms of cancer are ongoing. Highly important when developing novel vaccines are the safety aspects and also new adjuvants and delivery techniques needs to be carefully investigated so that they meet all short- and long-term safety requirements. One novel in vivo delivery method for plasmid vaccines is electroporation, which is the application of short pulses of electric current immediately after, and at the site of, an injection of a genetic vaccine. This method has been shown to significantly augment the transfection efficacy and the subsequent vaccine-specific immune responses. However, the dramatic increase in delivery efficacy offered by electroporation has raised concerns of potential increase in the risk of integration of plasmid DNA into the host genome. Here, we demonstrate the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine delivered intradermally using the needle free device Biojector 2000 together with electroporation using Derma Vax™ DNA Vaccine Skin Delivery System. We demonstrate that plasmids persist in the skin at the site of injection for at least four months after immunization. However, no association between plasmid DNA and genomic DNA could be detected as analyzed by qPCR following field inversion gel electrophoresis separating heavy and light DNA fractions. We will shortly initiate a phase I clinical trial in which healthy volunteers will be immunized with this multiplasmid HIV-1 vaccine using a combination of the delivery methods jet-injection and intradermal electroporation.

Enhancing efficacy and mucosa-tropic distribution of an oral HIV-PsV DNA vaccine in animal models.

A strategy combined the oral delivery route and bovine papillomavirus (BPV) pseudovirus (PsV)-based human immunodeficiency virus (HIV) DNA vaccine, which has been proven to enhance the mucosal immunization compared with the systemic immunization and in general does not induce effective mucosal immune responses. In this study, the immune responses against the BPV expressing HIV gp41 epitopes (ELDKWA, NWFDIT) after oral administration in Cynomolgus monkeys (Macaca fascicularis) were assessed, and the biodistribution of plasmid DNA encapsulated in the papillomavirus-like particles (VLPs) were evaluated in murine models. Results showed that oral immunization with the HIV-PsV DNA vaccine in monkey generated p24 and gp41 epitopes-specific serum IgG. Importantly, these induced antibodies had been shown to neutralize HIV-1 primary strain. In addition, the advantage of VLPs as vehicles delivering genes had been first revealed in biodistribution results. Therefore, orally administered HIV-PsV DNA vaccine was well-tolerated, enhanced the mucosa targeting property of the plasmid DNA, and reduced the nontargeting distribution, which indicate that it would reduce stress associated with systemic vaccination.

Absolute quantification of plasmid DNA by real-time PCR with genomic DNA as external standard and its application to a biodistribution study of an HIV DNA vaccine.

Real-time quantitative PCR (QPCR) has been proven to be a powerful tool for quantifying specific target DNA sequences. Compared to relative quantification, absolute quantification has the advantage of determining the absolute copy number of a given target, such as pathogen or plasmid DNA in vivo. However, matrix or impurities remaining in a DNA sample after various sample treatment procedures may influence a subsequent DNA analysis. In this work, we have compared methods of sample processing and validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of plasmid DNA in biological matrix, especially addressing the amplification inhibition due to matrix effect and sample complexity. Also, we applied our high-throughput sample preparation and absolute quantification method to determine the distribution of an HIV plasmid DNA vaccine in vivo. Successful application showed the validity and reliability of the method in absolute quantification of a particular gene in vivo.

In vivo biodistribution of a highly attenuated recombinant vesicular stomatitis virus expressing HIV-1 Gag following intramuscular, intranasal, or intravenous inoculation.

Recombinant vesicular stomatitis viruses (rVSVs) are being developed as potential HIV-1 vaccine candidates. To characterize the in vivo replication and dissemination of rVSV vectors in mice, high doses of a highly attenuated vector expressing HIV-1 Gag, rVSV(IN)-N4CT9-Gag1, and a prototypic reference virus, rVSV(IN)-HIVGag5, were delivered intramuscularly (IM), intranasally (IN), or intravenously (IV). We used quantitative, real-time RT-PCR (Q-PCR) and standard plaque assays to measure the temporal dissemination of these viruses to various tissues. Following IM inoculation, both viruses were detected primarily at the injection site as well as in draining lymph nodes; neither virus induced significant weight loss, pathologic signs, or evidence of neuroinvasion. In contrast, following IN inoculation, the prototypic virus was detected in all tissues tested and caused significant weight loss leading to death. IN administration of rVSV(IN)-N4CT9-Gag1 resulted in detection in numerous tissues (brain, lung, nasal turbinates, and lymph nodes) albeit in significantly reduced levels, which caused little or no weight loss nor any mortality. Following IV inoculation, both prototypic and attenuated viruses were detected by Q-PCR in all tissues tested. In contrast to the prototype, rVSV(IN)-N4CT9-Gag1 viral loads were significantly lower in all organs tested, and no infectious virus was detected in the brain following IV inoculation, despite the presence of viral RNA. These studies demonstrated significant differences in the biodistribution patterns of and the associated pathogenicity engendered by the prototypic and attenuated vectors in a highly susceptible host.

Cross-reactive HIV-1-neutralizing activity of serum IgG from a rabbit immunized with gp41 fused to IgG1 Fc: possible role of the prolonged half-life of the immunogen.

The elicitation of broadly cross-reactive HIV-1 neutralizing antibodies in humans remains a major challenge in developing a viable AIDS vaccine. We hypothesized that prolonged exposure to candidate vaccine immunogens could enhance the elicitation of such antibodies. In an attempt to develop HIV-1 vaccine immunogens with prolonged half-lives and increased stability, we constructed a fusion protein, gp41Fc, in which a truncated HIV-1 gp41(89.6) was fused to a human IgG(1) Fc. Gp41Fc is stable in solution, retains its antigenic structure and is highly immunogenic in rabbits. The serum titers reached 1:102,400 for the gp41Fc and 1:5,120 for gp140(89.6). Rabbit IgG neutralized diverse HIV-1 isolates and HIV-2, and the neutralization activity was attributed to gp41-specific IgG. The concentration of the gp41Fc in the serum correlated with the neutralization activity of rabbit IgG which recognized mostly conformation-independent epitopes on gp41 and predominantly bound to peptides derived from the gp41 immunodominant loop region. These results suggest that the prolonged half-life of gp41Fc in the serum may enhance the generation of cross-reactive neutralizing antibodies. Further research is needed to confirm and extend these results which may have implications for the development of vaccine immunogens with enhanced capability to elicit cross-reactive HIV-1-neutralizing antibodies.

Biodistribution and toxicological safety of adenovirus type 5 and type 35 vectored vaccines against human immunodeficiency virus-1 (HIV-1), Ebola, or Marburg are similar despite differing adenovirus serotype vector, manufacturer's construct, or gene inserts.

The Vaccine Research Center has developed vaccine candidates for different diseases/infectious agents (including HIV-1, Ebola, and Marburg viruses) built on an adenovirus vector platform, based on adenovirus type 5 or 35. To support clinical development of each vaccine candidate, pre-clinical studies were performed in rabbits to determine where in the body they biodistribute and how rapidly they clear, and to screen for potential toxicities (intrinsic and immunotoxicities). The vaccines biodistribute only to spleen, liver (Ad5 only), and/or iliac lymph node (Ad35 only) and otherwise remain in the site of injection muscle and overlying subcutis. Though approximately 10(11) viral particles were inoculated, already by Day 9, all but 10(3) to 10(5) genome copies per mu g of DNA had cleared from the injection site muscle. By three months, the adenovector was cleared with, at most, a few animals retaining a small number of copies in the injection site, spleen (Ad5), or iliac lymph node (Ad35). This pattern of limited biodistribution and extensive clearance is consistent regardless of differences in adenovector type (Ad5 or 35), manufacturer's construct and production methods, or gene-insert. Repeated dose toxicology studies identified treatment-related toxicities confined primarily to the sites of injection, in certain clinical pathology parameters, and in body temperatures (Ad5 vectors) and food consumption immediately post-inoculation. Systemic reactogenicity and reactogenicity at the sites of injection demonstrated reversibility. These data demonstrate the safety and suitability for investigational human use of Ad5 or Ad35 adenovector-based vaccine candidates at doses of up to 2 x 10(11) given intramuscularly to prevent various infectious diseases.

Towards biocompatible vaccine delivery systems: interactions of colloidal PECs based on polysaccharides with HIV-1 p24 antigen.

This work reports on the interactions of a model protein (p24, the capside protein of HIV-1 virus) with colloids obtained from polyelectrolyte complexes (PECs) involving two polysaccharides: chitosan and dextran sulfate (DS). The PECs were elaborated by a one-shot addition of default amounts of one counterpart to the polymer in excess. Depending on the nature of the excess polyelectrolyte, the submicrometric colloid was either positively or negatively charged. HIV-1 capsid p24 protein was chosen as antigen, the ultrapure form, lipopolysaccharide-free (endotoxin-, vaccine grade) was used in most experiments, as the level of purity of the protein had a great impact on the immobilization process. p24 sorption kinetics, isotherms, and loading capacities were investigated for positively and negatively charged particles of chitosans and dextran sulfates differing in degrees of polymerization (DP) or acetylation (DA). Compared with the positive particles, negatively charged colloids had higher binding capacities, faster kinetics, and a better stability of the adsorbed p24. Capacities up to 600 mg x g(-1) (protein-colloid) were obtained, suggesting that the protein interacted within the shell of the particles. Small-angle X-rays scattering experiments confirmed this hypothesis. Finally, the immunogenicity of the p24-covered particles was assessed for vaccine purposes in mice. The antibody titers obtained with immobilized p24 was dose dependent and in the same range as for Freund's adjuvant, a gold standard for humoral responses.

Oral immunization of rhesus macaques with adenoviral HIV vaccines using enteric-coated capsules.

Targeted delivery of vaccine candidates to the gastrointestinal (GI) tract holds potential for mucosal immunization, particularly against mucosal pathogens like the human immunodeficiency virus (HIV). Among the different strategies for achieving targeted release in the GI tract, namely the small intestine, pH sensitive enteric coating polymers have been shown to protect solid oral dosage forms from the harsh digestive environment of the stomach and dissolve relatively rapidly in the small intestine by taking advantage of the luminal pH gradient. We developed an enteric polymethacrylate formulation for coating hydroxy-propyl-methyl-cellulose (HPMC) capsules containing lyophilized Adenoviral type 5 (Ad5) vectors expressing HIV-1 gag and a string of six highly-conserved HIV-1 envelope peptides representing broadly cross-reactive CD4(+) and CD8(+) T cell epitopes. Oral immunization of rhesus macaques with these capsules primed antigen-specific mucosal and systemic immune responses and subsequent intranasal delivery of the envelope peptide cocktail using a mutant cholera toxin adjuvant boosted cellular immune responses including, antigen-specific intracellular IFN-gamma-producing CD4(+) and CD8(+) effector memory T cells in the intestine. These results suggest that the combination of oral adenoviral vector priming followed by intranasal protein/peptide boosting may be an effective mucosal HIV vaccination strategy for targeting viral antigens to the GI tract and priming systemic and mucosal immunity.

Preclinical safety assessment of a DNA vaccine using particle-mediated epidermal delivery in domestic pig, minipig and mouse.

DNA vaccination involves the direct injection of genes coding for specific antigenic proteins. One technique known as particle-mediated epidermal delivery (PMED) is a practical approach for epidermal delivery and provides a strong immune response. An important aspect of the preclinical safety assessment of DNA vaccines is the selection of a pharmacologically relevant animal model for the assessment of antigen expression, optimization of delivery and formulation of the plasmid. This paper describes a comparative study of domestic pig, minipig and mouse in regard to local tolerance and antigen expression of HIV immunotherapeutic using PMED. Pig/minipig is considered a good model for the safety assessment of DNA vaccines due to the similarity to human skin. Local reactions were evaluated at 10 min, 4, 24 and 48 h. Histology of administration sites revealed epidermal necrosis with associated dermal inflammation at 10 min and 4h, and subsequent regeneration with repair at 24 and 48 h. The degree and extent of these changes varied according to species. Domestic pig and minipig showed superficial epidermal necrosis and complete repair, while the mouse showed full-thickness epidermal necrosis and partial repair. Expression of HIV antigen was confirmed using immunohistochemistry in all three species at 4, 24 and 48 h. The results showed that PMED is an effective system for DNA vaccine delivery as demonstrated by the antigen expression seen as early as 4 h.

Biodistribution and persistence of an MVA-vectored candidate HIV vaccine in SIV-infected rhesus macaques and SCID mice.

Recombinant modified vaccinia virus Ankara (MVA) is together with a few other attenuated viral vectors on the forefront of human immunodeficiency virus type 1 (HIV-1) vaccine development. As such, MVA-vectored vaccines are likely to be administered into immunocompromized individuals. Here, we demonstrated in a good laboratory practice study safety and biological clearance of candidate HIV-1 vaccine MVA.HIVA in simian immunodeficiency virus (SIV)-infected rhesus macaques and mice with a severe combined immunodeficiency (SCID) following an intradermal vaccine administration. In SIV-infected macaques, MVA.HIVA DNA was undetectable by nested PCR 6 weeks after dosing. In SCID mice, the MVA.HIVA vaccine was well tolerated and a positive PCR signal was only observed at the site of injection 49 days after dosing in four out of six mice, but even these sites were negative by day 81 post-injection. Therefore, the MVA.HIVA vaccine is considered safe for application in phase I clinical trials in HIV-1-infected human subjects. These results also contribute to the confidence of using MVA as a smallpox vaccine.