Vaccination with a human parainfluenza virus type 3 chimeric FHN glycoprotein formulated with a combination adjuvant induces protective immunity.

Human parainfluenza virus type 3 (PIV3) is a major cause of lower respiratory disease i.e. bronchitis, bronchiolitis or pneumonia, in infants and young children. Presently there is no licensed vaccine against PIV3. To produce an effective subunit vaccine, a chimeric FHN glycoprotein consisting of the N-terminal ectodomain of the fusion (F) protein linked to the haemagglutinin-neuraminidase (HN) protein without transmembrane domain, and secreted forms of the individual F and HN glycoproteins, were expressed in mammalian cells and purified. Mice and cotton rats were immunized intramuscularly (IM) with FHN or both F and HN proteins (F + HN), formulated with poly(I:C) and an innate defense regulator peptide in polyphosphazene (TriAdj). Significantly higher levels of systemic virus-neutralizing antibodies were observed in mice and cotton rats immunized with FHN/TriAdj when compared to animals immunized with the combination of F and HN proteins (F + HN/TriAdj). As PIV3 is a pneumotropic virus, another goal is to produce an effective mucosal subunit vaccine. Intranasal (IN) administration with FHN/TriAdj resulted in mucosal IgA production in the lung and virus neutralizing antibodies in the sera. After PIV3 challenge no virus was detected in cotton rats immunized with FHN/TriAdj regardless of the route of delivery. Protective immunity against PIV3 was also induced by FHN/TriAdj in hamsters. In conclusion, the FHN protein formulated with TriAdj has potential for development of a safe and effective vaccine against PIV3.

Recombinant mumps viruses expressing the batMuV fusion glycoprotein are highly fusion active and neurovirulent.

A recent study reported the detection of a bat-derived virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV) with phylogenetic relatedness to human mumps virus (hMuV). Since all efforts to isolate infectious batMuV have reportedly failed, we generated recombinant mumps viruses (rMuVs) in which the open reading frames (ORFs) of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of an hMuV strain were replaced by the corresponding ORFs of batMuV. The batMuV F and HN proteins were successfully incorporated into viral particles and the resultant chimeric virus was able to mediate infection of Vero cells. Distinct differences were observed between the fusogenicity of rMuVs expressing one or both batMuV glycoproteins: viruses expressing batMuV F were highly fusogenic, regardless of the origin of HN. In contrast, rMuVs expressing human F and bat-derived HN proteins were less fusogenic compared to hMuV. The growth kinetics of chimeric MuVs expressing batMuV HN in combination with either hMuV or batMuV F were similar to that of the backbone virus, whereas a delay in virus replication was obtained for rMuVs harbouring batMuV F and hMuV HN. Replacement of the hMuV F and HN genes or the HN gene alone by the corresponding batMuV genes led to a slight reduction in neurovirulence of the highly neurovirulent backbone strain. Neutralizing antibodies inhibited infection mediated by all recombinant viruses generated. Furthermore, group IV anti-MuV antibodies inhibited the neuraminidase activity of bat-derived HN. Our study reports the successful generation of chimeric MuVs expressing the F and HN proteins of batMuV, providing a means for further examination of this novel batMuV.

Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3 vector in chickens.

Newcastle disease virus (NDV) belongs to serotype 1 of the avian paramyxoviruses (APMV-1) and causes severe disease in chickens. Current live attenuated NDV vaccines are not fully satisfactory. An alternative is to use a viral vector vaccine that infects chickens but does not cause disease. APMV serotype 3 infects a wide variety of avian species but does not cause any apparent disease in chickens. In this study, we constructed a reverse-genetics system for recovery of infectious APMV-3 strain Netherlands from cloned cDNAs. Two recombinant viruses, rAPMV3-F and rAPMV3-HN, were generated expressing the NDV fusion (F) and hemagglutinin-neuraminidase (HN) proteins, respectively, from added genes. These viruses were used to immunize 2-week-old chickens by the oculonasal route in order to evaluate the contribution of each protein to the induction of NDV-specific neutralizing antibodies and protective immunity. Each virus induced high titers of NDV-specific hemagglutination inhibition and serum neutralizing antibodies, but the response to F protein was greater. Protective immunity was evaluated by challenging the immunized birds 21 days later with virulent NDV via the oculonasal, intramuscular, or intravenous route. With oculonasal or intramuscular challenge, all three recombinant viruses (rAPMV3, rAPMV3-F, and rAPMV3-HN) were protective, while all unvaccinated birds succumbed to death. These results indicated that rAPMV3 alone can provide cross-protection against NDV challenge. However, with intravenous challenge, birds immunized with rAPMV3 were not protected, whereas birds immunized with rAPMV3-F alone or in combination with rAPMV3-HN were completely protected, and birds immunized with rAPMV3-HN alone were partially protected. These results indicate that the NDV F and HN proteins are independent neutralization and protective antigens, but the contribution by F is greater. rAMPV3 represents an avirulent vaccine vector that can be used against NDV and other poultry pathogens.

Positive selection in the hemagglutinin-neuraminidase gene of Newcastle disease virus and its effect on vaccine efficacy.

BACKGROUND: To investigate the relationship between the selective pressure and the sequence variation of the hemagglutinin-neuraminidase (HN) protein, we performed the positive selection analysis by estimating the ratio of non-synonymous to synonymous substitutions with 132 complete HN gene sequences of Newcastle disease viruses (NDVs) isolated in China. RESULTS: The PAML software applying a maximum likelihood method was used for the analysis and three sites (residues 266, 347 and 540) in the HN protein were identified as being under positive selection. Codon 347 was located exactly in a recognized antigenic determinant (residues 345-353) and codon 266 in a predicted linear B-cell epitope. Substitutions at codon 540 contributed to the N-linked glycosylation potential of residue 538. To further evaluate the effect of positively selected sites on the vaccine efficacy, we constructed two recombinant fowlpox viruses rFPV-JS6HN and rFPV-LaSHN, expressing the HN proteins from a genotype VII field isolate Go/JS6/05 (with A266, K347 and A540) and vaccine strain La Sota (with V266, E347 and T540), respectively. Two groups of SPF chickens, 18 each, were vaccinated with the two recombinant fowlpox viruses and challenged by Go/JS6/05 at 3 weeks post-immunization. The results showed that rFPV-JS6HN could elicit more effective immunity against the prevalent virus infection than rFPV-LaSHN in terms of reducing virus shedding. CONCLUSIONS: The analysis of positively selected codons and their effect on the vaccine efficacy indicated that the selective pressure on the HN protein can induce antigenic variation, and new vaccine to control the current ND epidemics should be developed.

[Anti-tumor immunity of Newcastle disease virus HN protein is influenced by differential subcellular targeting].

BACKGROUND AND OBJECTIVE: Hemagglutinin-neuraminidase (HN) protein of newcastle disease virus is an important immunogen for oncolysis. We designed three different expression plasmids encoding the HN protein targeted to different subcellular compartments: cytoplasmic (Cy-HN), secreted (Sc-HN) and membrane-anchored (M-HN). On the basis of antitumor effect in vitro, the aim of this study is to investigate the anti-tumor immunity effect of HN protein in vivo. METHODS: In the present study, we developed a mouse model in order to evaluate the anti-tumor effect of the intratumorally injected modified HN proteins and the anti-tumor immunity by lymphocyte proliferative response and CTL activity test. RESULTS: Although all three DNA constructs elicited an immune response, tumor-bearing mice intratumorally injected with M-HN demonstrated a significantly better anti-tumor effect than those injected with Cy-HN or Sc-HN (Day 18: P=0.022; Day 21: P<0.01). It also showed that this anti-tumor effect was mediated by higher lymphocyte proliferative response and CTL activity in mice intratumorally injected with M-HN [M-HN vs Cy-HN, P=0.019; M-HN vs Sc-HN, P=0.043; M-HN vs pcDNA3.1(+), P<0.01]. CONCLUSION: The anti-tumor immunity of Newcastle disease virus HN protein is influenced by differential subcellular targeting. The membrane-anchored form of the HN protein appears to be an ideal candidate to improve the specific cellular immunity.

IgA immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection.

IgA is considered to be the principal Ab involved in defense against pathogens in the mucosal compartment. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have examined the precise role of IgA in protective anti-influenza responses after intranasal vaccination. IgA(-/-) mice immunized intranasally with soluble hemagglutinin (hemagglutinin subtype 1) and neuraminidase (neuraminidase subtype 1) vaccine in the absence of adjuvant were found to be more susceptible to influenza virus infection than IgA(+/+) mice (13 vs 75% survival after virus challenge). Inclusion of IL-12 during immunization restored the protective efficacy of the vaccine to that seen in IgA(+/+) animals. IgA(-/-) mice had no detectable IgA expression, but displayed enhanced serum and pulmonary IgM and IgG Ab levels after IL-12 treatment. Assessment of T cell function revealed markedly depressed splenic lymphoproliferative responses to PHA in IgA(-/-) animals compared with IgA(+/+) mice. Furthermore, IgA(-/-) animals displayed impaired T cell priming to the H1N1 subunit vaccine, with concomitant reduction in recall memory responses due to a defect in APC function. Collectively, these results provide evidence that a major role of IgA is to facilitate presentation of Ag to mucosal T cells. IL-12 treatment can overcome IgA deficiency by providing adequate T cell priming during vaccination.

CD4+ T cell priming accelerates the clearance of Sendai virus in mice, but has a negative effect on CD8+ T cell memory.

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421-436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.