Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013.

BACKGROUND: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry. RESULTS: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013. CONCLUSIONS: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.

A Recombinant Turkey Herpesvirus Expressing F and HN Genes of Avian Avulavirus-1 (AAvV-1) Genotype VI Confers Cross-Protection against Challenge with Virulent AAvV-1 Genotypes IV and VII in Chickens.

Newcastle disease (ND) is responsible for significant economic losses in the poultry industry. The disease is caused by virulent strains of Avian avulavirus 1 (AAvV-1), a species within the family Paramyxoviridae. We developed a recombinant construct based on the herpesvirus of turkeys (HVT) as a vector expressing two genes: F and HN (HVT-NDV-F-HN) derived from the AAvV-1 genotype VI ("pigeon variant" of AAvV-1). This recombinant viral vaccine candidate was used to subcutaneously immunize one group of specific pathogen-free (SPF) chickens and two groups of broiler chickens (20 one-day-old birds/group). Humoral immune response was evaluated by hemagglutination-inhibition test and enzyme-linked immunosorbent assay (ELISA). The efficacy of the immunization was assessed in two separate challenge studies performed at 6 weeks of age with the use of virulent AAvV-1 strains representing heterologous genotypes IV and VII. The developed vaccine candidate elicited complete protection in SPF chickens since none of the birds became sick or died during the 2-week observation period. In the broiler groups, 90% and 100% clinical protection were achieved after challenges with AAvV-1 of IV and VII genotypes, respectively. We found no obvious relationship between antibody levels and protection assessed in broilers in the challenge study. The developed recombinant HVT-NDV-F-HN construct containing genes from a genotype VI AAvV-1 offers promising results as a potential vaccine candidate against ND in chickens.

A recombinant adenovirus expressing the E protein of duck Tembusu virus induces protective immunity in duck.

Duck Tembusu virus disease, caused by the duck Tembusu virus (DTMUV), can lead to a severe reduction in egg production and growth retardation in laying ducks and ducklings, respectively. In this study, we engineered a novel recombinant adenovirus expressing the E protein of DTMUV (rAd-E) in AAV-293 cells (analyzed by western blot and indirect immunofluorescence assays). Intramuscular immunization of Cherry Valley ducks with rAd-E was performed to evaluate host cellular and humoral immune responses. Compared to the phosphate-buffered saline administered group and the negative control wild-type adenovirus (wtAd) group, the rAd-E vaccinated group showed increased cellular and humoral responses. The results from the cytokine release and lymphocyte proliferation assays showed that rAd-E induced a stronger cellular immune response than the control group (P<0.01), 4 weeks after primary immunization. The results of enzyme-linked immunosorbent and virus neutralization assays showed that rAd-E induced higher titers of specific neutralizing antibodies, 2 weeks after primary immunization. The DTMUV challenge experiment showed a higher survival rate (80%) of ducks in the rAd-E group, when challenged with 0.5 ml (ELD50=10-2.67/0.2 ml) of the DTMUV strain AH-F10. These results indicate that rAd-E effectively protects ducks against DTMUV infection. Therefore, rAd-E could be a vaccine candidate to provide an effective and safe method for prevention and control of DTMUV infection.

Serotype-specific Binding Properties and Nanoparticle Characteristics Contribute to the Immunogenicity of rAAV1 Vectors.

The immunogenic properties of recombinant adeno-associated virus (rAAV) gene transfer vectors remain incompletely characterized in spite of their usage as gene therapy vectors or as vaccines. Molecular interactions between rAAV and various types of antigen-presenting cells (APCs), as well as the impact of these interactions on transgene or capsid-specific immunization remain unclear. We herein show that binding motifs recognized by the capsid and which determine the vector tissue tropism are also critical for key immune activation processes. Using rAAV capsid serotype 1 (rAAV1) vectors which primary receptors on target cells are α2,3 and α2,6 N-linked sialic acids, we show that sialic acid-dependent binding of rAAV1 on APCs is essential to trigger CD4(+) T-cell responses by increasing rAAV1 uptake and contributing to antigenic presentation of both the capsid and transgene product although this involves different APCs. In addition, the nanoparticulate structure of the vector in itself appears to be sufficient to trigger mobilization and activation of some APCs. Therefore, combinations of structural and of serotype-specific cell-targeting properties of rAAV1 determine its complex immunogenicity. These findings may be useful to guide a selection of rAAV variants depending on the intended level of immunogenicity for either gene therapy or vaccination applications.

Oral attenuated Salmonella enterica serovar Typhimurium vaccine expressing codon-optimized HIV type 1 Gag enhanced intestinal immunity in mice.

Oral immunization is a safe and easily applicable route to induce mucosal immunity to HIV infection. We examined the ability of oral attenuated Salmonella typhimurium (ST) vaccine expressing Gag for the efficiency of generating Gag-specific mucosal IgA and CD8+ T cell responses in intestinal lymphoid tissues. By optimizing the codon of HIV-1 gag to the preferred codon bias of Salmonella, the expression of Gag in Salmonella was dramatically improved. The oral ST-Gag vaccine by itself was not so powerful and induces little Gag-specific CD8+ T cell responses in the intestine. Nevertheless, we found that it potentiates otherwise weak intestinal CD8+ T cell responses in nasally primed mice with Gag p24 and cholera toxin adjuvant. Thus, the oral delivery of Salmonella expressing Gag would be utilized in combination with other parenteral vaccine to direct and strengthen intestinal HIV-specific CTL responses.

Recombinant canarypox vectored West Nile virus (WNV) vaccine protects dogs and cats against a mosquito WNV challenge.

The safety and efficacy of a canarypox vector expressing PrM and E genes of West Nile virus (WNV) (ALVAC-WNV) was evaluated in dogs and cats. One group of 17 dogs (vaccinated with 10(5.6) TCID(50)) and two groups of cats (groups 1 [n=14] vaccinated with 10(7.5) TCID(50) and 2 [n=8] 10(5.6) TCID(50)) were vaccinated twice at 28-day intervals. Fifteen dogs and eleven cats served as negative controls. The cats and dogs were challenged 120 and 135 days after the second immunization, respectively via the bites of Aedes albopictus mosquitoes infected with WNV. The first dose of vaccine induced a detectable antibody response in four dogs and five cats (one immunized with low and four with high doses). After the second dose, all the vaccinated dogs and all of the cats, immunized with high dose had detectable antibody titers, whereas only four of eight cats in the low dose group were seropositive. None of the vaccinated dogs and one vaccinated cat developed viremia following the WNV mosquito-challenge. In contrast, 14 of the 15 control dogs and 9 of the 11 control cats developed viremia. The experimental vaccine described in this study may be of value in the prevention of WNV infection in dogs and cats.

Primary replication of a recombinant Sendai virus vector in macaques.

An efficient antigen expression system using a recombinant Sendai virus (SeV) has been established recently and its potential to induce resistance against immunodeficiency virus infections in macaques has been shown. SeV replication has been well characterized in mice, the natural host, but not in primates, including humans. Here, primary SeV replication was investigated in macaques. After intranasal immunization with a recombinant SeV expressing simian immunodeficiency virus Gag protein, SeV-Gag, robust gag expression was observed in the nasal mucosa and much lower but significant levels of gag expression were observed in the local retropharyngeal and submandibular lymph nodes (LN). Expression peaked within a week and lasted at least up to 13 days after immunization. SeV-Gag was isolated from nasal swabs consistently at day 4 but not at all at day 13. Gag expression was undetectable in the lung as well as in remote lymphoid tissues, such as the thymus, spleen and inguinal LN, indicating that the spread of the virus was more restricted in macaques than in mice. SeV-specific T cells were detectable in SeV-immunized macaques at day 7. Finally, no naive macaques showed significant levels of anti-SeV antibodies in the plasma, even after living in a cage together with an acutely SeV-infected macaque for 5 weeks, indicating that SeV transmission from SeV-infected macaques to naive ones was inefficient. None of the SeV-immunized macaques displayed appreciable clinical manifestations. These results support the idea that this system may be used safely in primates, including humans.

B16 as a mouse model for human melanoma.

This unit details protocols for in vivo models of subcutaneous growth and pulmonary metastases of B16 melanoma. Therapeutic approaches include the use of B16.GM-CSF and rVVmTRP-1 to induce autoimmune vitiligo and tumor protection. The induction and use of gp 100-specific therapeutic cytotoxic T lymphocytes (CTL) are discussed. Methods are also included for CTL induction, isolation and testing, CTL maintenance, and adoptive transfer. Support protocols detail the testing of mouse sera for presence of MDA-specific antibodies by immunoblotting and ELISA, respectively. Additional sections, including growing B16 melanoma, enumerating pulmonary metastases, and use of recombinant viruses for vaccination, are discussed together with safety concerns.