In Depth Breadth Analyses of Human Blockade Responses to Norovirus and Response to Vaccination.

To evaluate and understand the efficacy of vaccine candidates, supportive immunological measures are needed. Critical attributes for a norovirus vaccine are the strength and breadth of antibody responses against the many different genotypes. In the absence of suitable neutralization assays to test samples from vaccine clinical trials, blockade assays offer a method that can measure functional antibodies specific for many of the different norovirus strains.

Immunogenicity and specificity of norovirus Consensus GII.4 virus-like particles in monovalent and bivalent vaccine formulations.

Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E.

H1N1 influenza virus-like particles: physical degradation pathways and identification of stabilizers.

A simple and rapid approach to vaccine stabilization has been applied to a novel virus-like particle (VLP) that contains the primary influenza antigens (hemagglutinin and neuraminidase surface proteins). A complement of spectroscopic and light scattering techniques was used to characterize the physical stability of influenza VLPs as a function of temperature and pH, two pharmaceutically relevant stress factors. The resulting data set was mathematically converted into a three-color empirical phase diagram (EPD) that illustrates changes in physical state as a function of these stress factors.

Influenza virus-like particle vaccines.

Enveloped virus-like particle (VLP) vaccines containing influenza hemagglutinin (HA) and neuraminidase (NA) antigens are produced easily in insect or mammalian cells via the simultaneous expression of HA and NA along with a viral core protein, such as influenza matrix (M1) or a retroviral Gag protein. The size and shape of the resulting particles are dictated by the choice of the core component, but M1- and Gag-based VLPs are strongly immunogenic and protective in seasonal and highly pathogenic influenza challenge models. Current data are consistent with the hypothesis that influenza VLP vaccine efficacy is related to the particulate, multivalent composition coupled with the presence of correctly folded antigens with intact biological activities.

Influenza-pseudotyped Gag virus-like particle vaccines provide broad protection against highly pathogenic avian influenza challenge.

Influenza-pseudotyped Gag virus-like particles (VLPs) were produced via the expression of influenza hemagglutinin (HA), neuraminidase (NA) and the murine leukemia virus Gag product in the baculovirus-insect cell expression system. Hemagglutination specific activities of sucrose gradient-purified VLPs were similar to those of egg-grown influenza viruses but particle morphologies were gamma retrovirus-like in the form of consistent 100nm spheres. Immunization of mice and ferrets demonstrated robust immunogenicity and protection from challenge with no measurable morbidity.

Potent protective cellular immune responses generated by a DNA vaccine encoding HSV-2 ICP27 and the E. coli heat labile enterotoxin.

A mouse model was employed to evaluate protective cellular immune responses induced by an immediate early antigen of HSV-2. Particle-mediated DNA vaccination of mice with a DNA plasmid-encoding ICP27 resulted in the induction of ICP27-specific IFN-gamma and TNF-alpha production in Balb/c mice, but little protection to intranasal challenge with wild type HSV-2. However, when the DNA vaccine was supplemented with as little as 50ng of a vector encoding the A and B subunits of the Escherichia coli heat labile enterotoxin (LT), animals were profoundly protected from morbidity and mortality.

DNA immunization in combination with effective antiretroviral drug therapy controls viral rebound and prevents simian AIDS after treatment is discontinued.

DNA immunization in conjunction with antiretroviral therapy was evaluated in SIV-infected rhesus macaques treated with [R]-9-[2-phosphonylmethoxypropyl]adenine (PMPA). Macaques were immunized monthly with DNA vaccines expressing either SIV gag/tat or SIV gag/tat and 19 CD8+ T cell epitopes during 7 months of therapy. Half the animals from each group were additionally immunized before infection.

Epidermal DNA vaccine for influenza is immunogenic in humans.

A phase I clinical trial was conducted to evaluate a monovalent influenza DNA vaccine containing the HA gene from A/Panama/2007/99 delivered by particle-mediated epidermal delivery (PMED). Three groups of 12 healthy adult subjects received a single dose on day 0 of either 1, 2 or 4 microg of DNA vaccine, delivered as 1, 2 or 4 PMED administrations. The PMED influenza DNA vaccine elicited serum hemagglutination-inhibition (HAI) antibody responses at all three dose levels, with the highest and most consistent responses in subjects vaccinated with the highest dose level.

The role of particle-mediated DNA vaccines in biodefense preparedness.

Particle-mediated epidermal delivery (PMED) of DNA vaccines is based on the acceleration of DNA-coated gold directly into the cytoplasm and nuclei of living cells of the epidermis, facilitating DNA delivery and gene expression. Professional antigen-presenting cells and keratinocytes in the skin are both targeted, resulting in antigen presentation via direct transfection and cross-priming mechanisms. Only a small number of cells need to be transfected to elicit humoral, cellular and memory responses, requiring only a low DNA dose.

Particle-mediated DNA vaccine delivery to the skin.

Particle-mediated DNA vaccines employ a physical, intracellular delivery device to achieve the deposition of plasmid DNA-based expression vectors directly into the interior of cells of the skin. The resultant bolus of transient antigen expression in keratinocytes and trafficking dendritic cells results in the induction of humoral and cellular immune responses in various animal models and humans, mimicking characteristics of live or live-vectored vaccines. Ultimately, DNA vaccine success in the clinic will depend on both the successful intracellular delivery of a plasmid vector and an immunostimulator or adjuvant to maximise humoral and cellular immune responses to the encoded antigen(s).

Needle-free epidermal powder immunization.

Due to the presence of a network of antigen-presenting cells and other cells with innate and adaptive immune functions, the skin is both a sensitive immune organ and a practical target site for vaccine administration. A handful of needle-free immunization technologies have emerged in recent years that aim to take advantage of these characteristics. Skin delivery technologies provide potentially safer alternatives to needle injection and promises increased efficacy in the prevention and/or therapy of infectious diseases, allergic disorders and cancer.

Particle-mediated DNA vaccination of mice, monkeys and men: looking beyond the dogma.

Data generated in the early phases of experimentation in a new field of scientific exploration sometimes results in hasty conclusions about the generality of the data. To some degree, this has happened at least twice in the relatively new area of DNA immunization. Early data seemed to indicate firstly that particle-mediated epidermal DNA immunization induced predominantly Th2-type cellular immune responses, and secondly that DNA immunization was not very successful in humans.

Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines.

Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-gamma]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent.

Induction of mucosal protection against primary, heterologous simian immunodeficiency virus by a DNA vaccine.

An effective vaccine against human immunodeficiency virus (HIV) should protect against mucosal transmission of genetically divergent isolates. As a safe alternative to live attenuated vaccines, the immunogenicity and protective efficacy of a DNA vaccine containing simian immunodeficiency virus (SIV) strain 17E-Fr (SIV/17E-Fr) gag-pol-env was analyzed in rhesus macaques. Significant levels of cytotoxic T lymphocytes (CTL), but low to undetectable serum antibody responses, were observed following multiple immunizations.

Development of a Genetically-Engineered, Candidate Polio Vaccine Employing the Self-Assembling Properties of the Tobacco Mosaic Virus Coat Protein.

A synthetic gene coding for the coat protein of tobacco mosaic virus (TMVCP) was expressed in under the direction of the UV5 promoter. Modification of the 3' end of the TMVCP gene by insertion of a region coding for an antigenic epitope from poliovirus type 3 resulted in the production of a hybrid TMVCP (TMVCP-polio 3). Both the -produced TMVCP and TMVCP-polio 3 were shown to assemble into virus-like rods under acidic conditions in extracts.