DNA vaccination against influenza viruses: a review with emphasis on equine and swine influenza

The influenza virus vaccines that are commercially-available for humans, horses and pigs in the United States are inactivated, whole-virus or subunit vaccines. While these vaccines may decrease the incidence and severity of clinical disease, they do not consistently provide complete protection from virus infection. DNA vaccines are a novel alternative to conventional vaccination strategies, and offer many of the potential benefits of live virus vaccines without their risks. In particular, because immunogens are synthesized de novo within DNA transfected cells, antigen can be presented by MHC class I and II molecules, resulting in stimulation of both humoral and cellular immune responses. Influenza virus has been used extensively as a model pathogen in DNA vaccine studies in mice, chickens, ferrets, pigs, horses and non-human primates, and clinical trials of DNA-based influenza virus vaccines are underway in humans. Our studies have focused on gene gun delivery of DNA vaccines against equine and swine influenza viruses in mice, ponies and pigs, including studies employing co-administration of interleukin-6 DNA as an approach for modulating and adjuvanting influenza virus hemagglutinin-specific immune responses. The results indicate that gene gun administration of plasmids encoding hemagglutinin genes from influenza viruses is an effective method for priming and/or inducing virus-specific immune responses, and for providing partial to complete protection from challenge infection in mice, horses and pigs. In addition, studies of interleukin-6 DNA co-administration in mice clearly demonstrate the potential for this approach to enhance vaccine efficacy and protection.     

Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses

Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes (A/sw/Potsdam/15/1981 H1N1, A/sw/Bad Griesbach/IDT5604/2006 H1N1, A/sw/Bakum/1832/2000 H1N2, A/sw/Damme/IDT5673/2006 H3N2, A/sw/Herford/IDT5932/2007 H3N2). The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses.     

Evaluation of the ability of canarypox-vectored equine influenza virus vaccines to induce humoral immune responses against canine influenza viruses in dogs

OBJECTIVE: To evaluate canarypox-vectored equine influenza virus (EIV) vaccines expressing hemagglutinins of A/equine/Kentucky/94 (vCP1529) and A2/equine/Ohio /03 (vCP2242) for induction of antibody responses against canine influenza virus (CIV) in dogs. ANIMALS: 35 dogs. PROCEDURES: Dogs were randomly allocated into 4 groups; group 1 (n = 8) and group 2 (9) were inoculated SC on days 0 and 28 with 1.0 mL (approx 10(5.7) TCID(50)) of vCP1529 and vCP2242, respectively. Dogs in group 3 (n = 9) were inoculated twice with 0.25 mL (approx 10(5.7) TCID(50)) of vCP2242 via the transdermal route. The 9 dogs of group 4 were control animals. All dogs were examined for adverse reactions. Sera, collected on days -1, 7, 13, 21, 28, 35, and 42, were tested by hemagglutination inhibition (HI) and virus neutralization (VN) assays for antibodies against CIV antigens A/Canine/FL/43/04-PR and A/Canine/NY/115809/05, respectively. RESULTS: Inoculations were tolerated well. The HI and VN antibodies were detected by 7 days after primary inoculation. Most dogs of groups 1 and 2 and all dogs of group 3 had detectable antibodies by 14 days after initial inoculation. The second inoculation induced an anamnestic response, yielding geometric mean HI titers of 139, 276, and 1,505 and VN titers of 335, 937, and 3,288 by day 42 (14 days after booster inoculation) in groups 1, 2, and 3, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Canarypox-vectored EIV vaccines induce biologically important antibodies and may substantially impact CIV transmission within a community and be of great value in protecting dogs against CIV-induced disease.     

Phylogenetic analysis of swine influenza viruses isolated in Poland

Swine influenza virus (SIV) of H1N1 and H3N2 subtypes are dominated in European pigs population. "Classical swine" H1N1 subtype was replaced by "avian-like" H1N1 subtype. It co-circulates with H3N2 reassortant possessing "avian" genes. In the present study, 41 SIV strains isolated from pigs with pneumonia, raised in 20 Polish farms, were identified and characterised. Since it was evidenced that isolates from the same geographic district and the same year of isolation are in 100% similar, 15 strains representing different district and different year of isolation were chosen to construct phylogenetic trees. Two genes, conservative matrix 1 (M1) and the most variable, haemagglutynin (HA), were sequenced and subjected into phylogenetic analysis. The results of the analysis confirmed that "avian-like" swine H1N1 strains evolved faster than classical SIV strains. HA gene of these isolates have been derived from contemporary strains of "avian-like" SIV. In contrast, the M1 gene segment may have originated from avian influenza viruses. H3N2 strain is located in swine cluster, in the main prevalent European group of H3N2 isolates called A/Port Chalmers/1/73-like Eurasian swine H3N2 lineage, which has evolved separately from the human H3N2 virus lineage around 1973.     

基因重排H1N2亚型猪流感病毒的分离鉴定和生物学特性的研究

本研究对2005年～2006年广西和海南省疑似猪流感(SD)病猪的组织病料进行了病毒分离,并对分离毒株进行了亚型鉴定和生物学特性的研究.结果显示:分离的3株流感病毒均为H1N2亚型猪流感病毒(SIV).能凝集多种动物的红细胞,但凝集谱与以往报道略有不同;为热不稳定型病毒;在电镜下可观察到典型流感病毒粒子.动物试验显示:小白鼠、大白鼠和家兔对分离毒株不敏感,而豚鼠较敏感.能较好地复制出流感症状和病理变化;本体试验动物仅有轻微的临床症状,但能检测到抗体升高的变化,并能从鼻腔和上呼吸道检测和分离到SIV.核苷酸同源性分析显示:分离毒株Sw/GX/17/05、Sw/GX/13/06和Sw/HN/1/05的血凝素(HA)基因分别与基因重排H1N2 亚型SIV A/Swine/lndiana 9K035/99、A/SW/MN/23124-T/01和A/swine/Zhejiang/1/04的核苷酸同源性最高,分别达97.4%、97.0%和95.7%;神经氨酸酶(NA)基因均与基因重排H1N2 亚型流感病毒A/Trurkey/MO/24093/99 的核苷酸同源性最高,达97.2%～98.0%.核苷酸同源性分析进一步证实了分离毒株为基因重排H1N2亚型SIV.     

Antibody responses of horses to equine influenza viruses during a postepizootic period in Japan.

The antibody responses to equine influenza viruses were investigated during a postepizootic period of the disease. Serum samples were collected from a total of 128 horses on three occasions during the years 1967-77. No significant increase of hemagglutination-inhibition antibody titers to subtypes 1 and 2 of equine influenza virus were detected in any of the sera tested. The maternal hemagglutination-inhibition antibody titers of foals decreased over a four month interval. A marked increase of the titers was recognized in only the equine influenza virus vaccinated horses. These findings suggest that equine influenza virus was not prevalent in the horse populations during the observation period. In such conditions, the dissemination of equine influenza viruses in the horses is discussed in relation to introduction of the disease from abroad. We also examined whether the doctrine of original antigenic sin, an immunological phenomenon recognized in human influenza, was applicable for equine influenza. However, no marked increase of hemagglutination-inhibition antibody titer to the primary infecting subtype in the 44 horses was observed after administration of the heterologous subtype vaccine.     

Genomic characterization of H1N2 swine influenza viruses in Italy

Three subtypes (H1N1, H1N2, and H3N2) are currently diffused worldwide in pigs. The H1N2 subtype was detected for the first time in Italian pigs in 1998. To investigate the genetic characteristics and the molecular evolution of this subtype in Italy, we conducted a phylogenetic analysis of whole genome sequences of 26 strains isolated from 1998 to 2010. Phylogenetic analysis of HA and NA genes showed differences between the older (1998-2003) and the more recent strains (2003-2010). The older isolates were closely related to the established European H1N2 lineage, whereas the more recent isolates possessed a different NA deriving from recent human H3N2 viruses. Two other reassortant H1N2 strains have been detected: A/sw/It/22530/02 has the HA gene that is closely related to H1N1 viruses; A/sw/It/58769/10 is an uncommon strain with an HA that is closely related to H1N1 and an NA similar to H3N2 SIVs. Amino acid analysis revealed interesting features: a deletion of two amino acids (146-147) in the HA gene of the recent isolates and two strains isolated in 1998; the presence of the uncommon aa change (N66S), in the PB1-F2 protein in strains isolated from 2009 to 2010, which is said to have contributed to the increased virulence. These results demonstrate the importance of pigs as mixing vessels for animal and human influenza and show the presence and establishment of reassortant strains involving human viruses in pigs in Italy. These findings also highlighted different genomic characteristics of the NA gene the recent Italian strains compared to circulating European viruses.     

In vitro efficacies of oseltamivir carboxylate and zanamivir against equine influenza A viruses

To investigate the possibilities of two NA inhibitors [oseltamivir carboxylate (OC) and zanamivir (ZA)] as the clinical agents for equine influenza A virus (EIV) infection, we examined the efficacies of these inhibitors against twelve EIVs in vitro. OC and ZA inhibited NA activities of all EIVs with 50% inhibitory concentrations with ranging from 0.017 to 0.130 and from 0.010 to 0.074 microM, respectively. OC and ZA inhibited plaque-forming of all EIVs in MDCK cells with 50% effective concentrations with ranging from 0.015 to 0.097 and from 0.016 to 0.089 microM, respectively, except for one strain (13.328 microM and 6.729 microM). These results suggest that these inhibitors are effective against most EIVs and might be useful for treatment of EI in horses.     

Replication of a waterfowl-origin influenza virus in the kidney and intestine of chickens

Intravenous inoculation of chickens with a waterfowl-origin type A influenza virus resulted in high titers of virus in kidney tissues and viral nucleoprotein in renal tubular epithelial cells and in intestinal mucosal epithelial cells. Virus titers in kidneys of four of eight clinically normal chickens sampled on days 3 and 5 postinoculation (PI), one dead chicken on day 3 PI, and one dead chicken on day 7 PI exceeded 10(6) mean embryo infectious dose per gram of tissue. Using immunofluorescent and immunoperoxidase staining, viral nucleoprotein was identified in the cytoplasm and nucleus of tubular epithelial cells in kidneys and in nucleus of mucosal epithelial cells lining villi in the lower small intestine. Based on the low intravenous pathogenicity index for this virus (0.3) along with the high virus titers in kidney tissues and localization of viral antigen in kidney important site for replication of avian influenza (AI) virus of low pathogenicity. Recovery of type A influenza viruses from cloacal swabs could result from viral replication in kidneys as well as in the lower intestine and/or the bursa of Fabricius.     

Antibody responses of swine to type A influenza viruses in the most recent several years.

A serological survey was conducted on 4,080 swine sera collected for the years 1985-90. The swine sera positive to A/New Jersey/8/76 (swine type H1N1) strain were observed in annual (10-20%) and monthly (20-40%) incidences during the observation period except for occasional months. Antibodies to recent human H1N1 viruses in swine were recognized in relation to the human H1N1 influenza epidemics. Antibody responses of swine to human H3N2 strains appeared irrespective of human epidemics with the virus in the years 1985-87. However, in 1988 almost no antibodies to three human H3N2 isolates of 1983-88 were observed for this year except a few months though the human epidemic occurred in the area. Although in 1989-90 many swine had antibodies to the three strains in the percentage of 3 to 35, no antibody to the latest isolate, A/Hokkaido/20/89 (H3N2), was found for almost all the months of both years. These findings differed markedly from the possible relationship between the prevalence of H3N2 virus-antibodies in swine and the human influenza epidemics, which were described previously in many reports including our studies.     

多种亚型猪流感病毒混合感染的纯化和鉴定

为了研究多种亚型猪流感病毒混合感染后病毒的纯化,试验采用鸡胚终点稀释法结合特异血清中和法对可能混合多种亚型猪流感病毒的样品进行纯化,并对纯化结果进行HI和RT-PCR试验进行验证。结果表明:此种方法纯化病毒是可行的,不同代次不含外源病毒。     

Different evolutionary trends of swine H1N2 influenza viruses in Italy compared to European viruses

European H1N2 swine influenza viruses (EU H1N2SIVs) arose from multiple reassortment events among human H1N1, human H3N2, and avian influenza viruses. We investigated the evolutionary dynamics of 53 Italian H1N2 strains by comparing them with EU H1N2 SIVs. Hemagglutinin (HA) phylogeny revealed Italian strains fell into four groups: Group A and B (41 strains) had a human H1 similar to EU H1N2SIVs, which probably originated in 1986. However Group B (38 strains) formed a subgroup that had a two-amino acid deletion at positions 146/147 in HA. Group C (11 strains) contained an avian H1 that probably originated in 1996, and Group D (1 strain) had an H1 characteristic of the 2009 pandemic strain. Neuraminidase (NA) phylogeny suggested a series of genomic reassortments had occurred. Group A had an N2 that originated from human H3N2 in the late 1970s. Group B had different human N2 that most likely arose from a reassortment with the more recent human H3N2 virus, which probably occurred in 2000. Group C had an avian-like H1 combined with an N2 gene from one of EU H1N2SIVs, EU H3N2SIVs or Human H3N2. Group D was part of the EU H3N2SIVs clade. Although selection pressure for HA and NA was low, several positively selected sites were identified in both proteins, some of which were antigenic, suggesting selection influenced the evolution of SIV. The data highlight different evolutionary trends between European viruses and currently circulating Italian B strains and show the establishment of reassortant strains involving human viruses in Italian pigs.     

禽流感病毒和猪流感病毒——人兽共患病危险的新认识(续完)

上期回顾:上期介绍了禽流感病毒、猪流感病毒和人流感病毒各自的特点,流感病毒在其天然宿主中的致病机理。     

Avian and swine influenza viruses: our current understanding of the zoonotic risk

The introduction of swine or avian influenza (AI) viruses in the human population can set the stage for a pandemic, and many fear that the Asian H5N1 AI virus will become the next pandemic virus. This article first compares the pathogenesis of avian, swine and human influenza viruses in their natural hosts. The major aim was to evaluate the zoonotic potential of swine and avian viruses, and the possible role of pigs in the transmission of AI viruses to humans. Cross-species transfers of swine and avian influenza to humans have been documented on several occasions, but all these viruses lacked the critical capacity to spread from human-to-human. The extreme virulence of H5N1 in humans has been associated with excessive virus replication in the lungs and a prolonged overproduction of cytokines by the host, but there remain many questions about the exact viral cell and tissue tropism. Though pigs are susceptible to several AI subtypes, including H5N1, there is clearly a serious barrier to infection of pigs with such viruses. AI viruses frequently undergo reassortment in pigs, but there is no proof for a role of pigs in the generation of the 1957 or 1968 pandemic reassortants, or in the transmission of H5N1 or other wholly avian viruses to humans. The major conclusion is that cross-species transmission of influenza viruses per se is insufficient to start a human influenza pandemic and that animal influenza viruses must undergo dramatic but largely unknown genetic changes to become established in the human population.