Thermal Stabilization of Viral Vaccines in Low-Cost Sugar Films.

Most currently available vaccines, particularly live vaccines, require the cold chain, as vaccine efficacy can be significantly hampered if they are not stored in a temperature range of 2-8 °C at all times. This necessity places a tremendous financial and logistical burden on vaccination programs, particularly in the developing world. The development of thermally stable vaccines can greatly alleviate this problem and, in turn, increase vaccine accessibility worldwide. In this paper, we detail a simple and cost-effective method for stabilizing live vaccines that uses FDA-approved materials. To this end, we dried enveloped DNA (Herpes Simplex Virus type 2) and RNA (Influenza A virus) viral vaccines in a pullulan and trehalose mixture. The results of these studies showed that the live-attenuated HSV-2 vaccine retained its efficacy for at least 2 months of storage at 40 °C, while the inactivated influenza vaccine was able to retain its immunogenicity for at least 3 months of storage at 40 °C. This work presents a simple approach that allows thermo-sensitive vaccines to be converted into thermo-stable vaccines that do not require refrigeration, thus contributing to the improvement of vaccine deployment throughout the world.

Detection of Residual Proteins UL5 and UL29 Using a Targeted Proteomics Approach in HSV529, a Replication-Deficient HSV-2 Vaccine Candidate.

HSV529 is a replication defective human herpes simplex virus (HSV)-2 viral vaccine candidate in clinical development. An engineered cell line is required to support production of HSV529 by transgenic expression of the HSV-1 transcription factors UL5 (HELI) and UL29 (DNBI). These 2 genes have been deleted from the vaccine candidate to ensure replication deficiency, and the transgene products are thus impurities that must be monitored in the final product. Multiple reaction monitoring (MRM) is a mass spectrometry (MS) workflow that can be used to quickly develop targeted protein detection and quantitation methods. An MRM method was developed for detection of the HSV-1 proteins UL5 and UL29 based on results from nano-liquid chromatography-MS/MS protein analysis of HSV529 material. Sensitivity, specificity, and linearity of response for the MRM workflow were established using high-flow ultra-performance liquid chromatography coupled to a tandem quadrupole mass analyzer. Results show that residual UL5 and UL29 proteins can be detected in the HSV529 candidate, and that MRM analysis provides the appropriate sensitivity and specificity required for quantitation. The transition from nano-flow to ultra-performance driven chromatography was found to improve method robustness without compromising the sensitivity of the assay.

Towards a rational design of an asymptomatic clinical herpes vaccine: the old, the new, and the unknown.

The best hope of controlling the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) pandemic is the development of an effective vaccine. However, in spite of several clinical trials, starting as early as 1920s, no vaccine has been proven sufficiently safe and efficient to warrant commercial development. In recent years, great strides in cellular and molecular immunology have stimulated creative efforts in controlling herpes infection and disease. However, before moving towards new vaccine strategy, it is necessary to answer two fundamental questions: (i) why past herpes vaccines have failed? (ii) Why the majority of HSV seropositive individuals (i.e., asymptomatic individuals) are naturally "protected" exhibiting few or no recurrent clinical disease, while other HSV seropositive individuals (i.e., symptomatic individuals) have frequent ocular, orofacial, and/or genital herpes clinical episodes? We recently discovered several discrete sets of HSV-1 symptomatic and asymptomatic epitopes recognized by CD4(+) and CD8(+) T cells from seropositive symptomatic versus asymptomatic individuals. These asymptomatic epitopes will provide a solid foundation for the development of novel herpes epitope-based vaccine strategy. Here we provide a brief overview of past clinical vaccine trials, outline current progress towards developing a new generation "asymptomatic" clinical herpes vaccines, and discuss future mucosal "asymptomatic" prime-boost vaccines that could optimize local protective immunity.

Phase I dose-escalation study of a monovalent heat shock protein 70-herpes simplex virus type 2 (HSV-2) peptide-based vaccine designed to prime or boost CD8 T-cell responses in HSV-naïve and HSV-2-infected subjects.

This was a phase I study to assess the safety, tolerability, and immunogenicity of escalating doses of AG-702, a noncovalent complex of an HLA A*0201-restricted epitope in the glycoprotein B protein of herpes simplex virus type 2 (gB2) and truncated human constitutive heat shock protein 70. Similar vaccines have been immunogenic in animals. Three injections of 10 to 250 mug were administered intradermally to HLA A*0201-bearing subjects who were either herpes simplex virus type 2 (HSV-2)-infected or HSV uninfected. Sixty-two participants received the vaccine, 60 completed the protocol, and T-cell data were accrued for 56 subjects. The vaccine was safe and well tolerated. New or boosted responses to the HSV-2 CD8 epitope were not detected. Baseline responses to an epitope in virion proteins 13/14 were higher than responses to the gB2 epitope. A heat shock protein vaccine with an HSV-2 peptide appears to be safe at the doses studied in healthy adults with or without HSV infection. Modifications of the dose, adjuvant, route, schedule, or HSV antigen may be required to improve responses.

The VesiVax system: a method for rapid vaccine development.

The VesiVax system is based upon the concept that highly potent vaccines can be designed by engineering proteins that are capable of stably inserting themselves into liposomes. Such a nanoscale liposomal particle can then serve as an immunogen for vaccine development. The VesiVax vaccine technology platform is designed to make it relatively easy to engineer and produce new vaccines quickly. Vaccines based on the VesiVax system have been designed against the influenza virus and herpes simplex type 2 virus, the causative agents of the "flu" and genital herpes, respectively. Both vaccines have been tested in animal models and have demonstrated significant protective efficacy from challenge with lethal doses of virus. Assays of the immunological parameters suggest that both T and B cell responses can be elicited by VesiVax vaccines. The safety profile of the VesiVax vaccines is expected to be much better than that of vaccines prepared by conventional techniques. Taken together, the inherent flexibility of the VesiVax platform is expected to facilitate the rapid development of new vaccines which are effective at stimulating protective immune responses.

Degradation of Quillaja saponaria Molina saponins: loss of the protective effects of a herpes simplex virus 1 subunit vaccine.

Quillaja saponins (Q. saponins) are readily hydrolyzed at neutral pH to yield degraded deacylated saponins (DS-saponins). Degradation of Q. saponins resulted in some reduction of their capacity to elicit IgG1, IgG2a and IgG2b isotypes against the highly immunogenic envelope glycoprotein D (gD) from herpes simplex virus, type 1 (HSV-1). Addition to gD of a dose of DS-saponins tenfold higher than the original Q. saponins dose stimulated lower IgG2a and IgG2b titers than those obtained with gD alone or combined with native saponins. However, the IgG1 response was somewhat similar in all the groups. In contrast, Q. saponins' deacylation resulted in a significant reduction in both the production of HSV-1 neutralizing antibodies and survival rates after viral challenge. Vaccination with gD alone did not protect mice against a lethal challenge with HSV-1, while the addition of Q. saponins to gD resulted in protection against HSV-1. Vaccines containing partially deacylated saponins yielded lower survival rates, while vaccines containing DS-saponins did not protect mice against HSV-1. Increasing the dose of DS-saponins tenfold resulted in a marginal increase in protection. These results show that degradation of Q. saponins during storage can have a deleterious effect on vaccines' efficacies.