Role of maternal immunity in the protection of newborn ferrets against infection with a virulent influenza virus

Intranasal infection of newborn ferrets with a virulent strain of influenza virus invariably resulted in their deaths following virus replication to high titre in both lung and nasal turbinates (Collie et al., 1980). However, a similar challenge of newborn ferrets born to mothers immunized by infection with virulent or attenuated viruses resulted in complete protection; no virus replicated in their lungs and little or no virus was isolated from their nasal turbinates. Protection appeared to be antibody-mediated since it was sub-type-specific and milk-derived since newborn ferrets born to non-immune mothers but fostered onto immune mothers exhibited a similar level of protection to neonates born to and suckled by immune mothers.     

Heterotypic immunity to influenza in ferrets.

Heterotypic immunity to influenza virus in ferrets operated against heterotypic influenza viruses but not heterologous viruses. Contrary to prior reports, the protection conferred lasted for at least 18 months. This type of immunity limited virus shedding but did not prevent infection. These results suggest that this phenomenon could play a role in determining the severity of infections caused by type A influenza viruses in humans.     

Cellular response in humans following vaccination with gripax influenza virus

Cellular response of peripheral blood lymphocytes to influenza antigens was measured in a group of young nurse-student volunteers (17–24 years old), following vaccination with a formol-inactivated trivalent influenza vaccine (Gripax). Cord blood lymphocytes (controls) did not react with any of the antigens. This excluded the possibility of any nonspecific mitogenicity of viral antigens. Viability of the cells was indicated by their responsiveness to phytohemagglutinin (PHA). Prior to immunization antigenic recognition to circulating strains (A/England (H₃N₂) and B/Hong Kong) was found in about 44% of the vaccinees; recognition of the recent strain A/USSR (H₁N₁) was found in only 10.5%. Following vaccination, approximately 80% of the subjects exhibited cellular response to all three vaccine strains. This includes the negative subjects, who showed an approximate 70% rate of conversion. There was no correlation between the antibody state and cellular response prior to and following vaccination as gathered from matched data of each participant.     

Local and systemic immunity to influenza infections in ferrets.

To establish whether immunity to influenza infection in the ferret is local or systemic, two sites of challenge were utilized: the nose and the anatomically isolated tracheal pouch. Infection of either site did not spread to the other site, and challenge of either site resulted in seroconversion by 13 days. Simultaneous challenge of both sites 21 days after the primary infection revealed that prior infection of the pouch prevented subsequent reinfection of the pouch, but not infection of the nose. Thus, systemic immunity did not prevent the initiation of nasal influenza infection in the ferret. However, the duration of virus shedding from the nose was reduced to half of that seen when ferrets were infected for the first time, showing that the prior pouch infection did lead to a more rapid recovery from the subsequent nasal infection. Passively administered anti-influenza antibody did not prevent or modify the nasal infection, but it did prevent the pouch infection. This is consistent with the observation that an initial infection of the nose prevented pouch infection upon challenge 21 days later. The prior nasal infection also prevented the subsequent nasal infection. These data suggest that immunity to acquisition of influenza infection in the ferret is a local phenomenon, whereas recovery from active infection is influenced by systemic immune mechanisms.     

Bacterial sinusitis and otitis media following influenza virus infection in ferrets

Streptococcus pneumoniae is the leading cause of otitis media, sinusitis, and pneumonia. Many of these infections result from antecedent influenza virus infections. In this study we sought to determine whether the frequency and character of secondary pneumococcal infections differed depending on the strain of influenza virus that preceded bacterial challenge. In young ferrets infected with influenza virus and then challenged with pneumococcus, influenza viruses of any subtype increased bacterial colonization of the nasopharynx. Nine out of 10 ferrets infected with H3N2 subtype influenza A viruses developed either sinusitis or otitis media, while only 1 out of 11 ferrets infected with either an H1N1 influenza A virus or an influenza B virus did so. These data may partially explain why bacterial complication rates are higher during seasons when H3N2 viruses predominate. This animal model will be useful for further study of the mechanisms that underlie viral-bacterial synergism.     

Enhancement of anti-influenza A virus cytotoxicity following influenza A virus vaccination in older, chronically ill adults.

We studied anti-influenza cytotoxicity by bulk peripheral blood mononuclear leukocyte (PBL) cultures derived from older, chronically ill volunteers undergoing vaccination. Vaccinees received either cold-recombinant, live-attenuated influenza A/Korea/1/82 (H3N2) virus intranasally or inactivated monovalent influenza A/Taiwan/1/86 (H1N1) subvirion vaccine intramuscularly. PBL were collected pre- and postvaccination and in vitro stimulated by autologous PBL infected with influenza A virus homologous and heterosubtypic to the respective vaccine strain. Cytotoxicity was measured against influenza A virus-infected autologous and human leukocyte antigen (HLA)-mismatched PBL targets infected with influenza A virus homologous or heterosubtypic to the vaccine virus strain. Vaccinees infected with the live-attenuated virus developed significant rises in mean anti-influenza, HLA-restricted cytotoxicity that was cross-reactive against influenza A viruses homologous and heterosubtypic to the vaccine virus. The enhanced cross-reactive cytotoxicity was inducible postvaccination by in vitro stimulation with autologous PBL infected with the homologous influenza A (H3N2) virus and with influenza A (H1N1) virus. In contrast, after vaccination with inactivated monovalent subvirion vaccine, volunteers developed significant increases in mean anti-influenza, HLA-restricted cytotoxicity only against autologous PBL infected with homologous influenza A (H1N1) virus. Increased cytotoxicity occurred only after in vitro stimulation with autologous cells infected with homologous influenza A (H1N1) virus. Mean gamma interferon levels in supernatant fluids of influenza A virus-stimulated effector PBL did not increase postvaccination, despite increased levels of anti-influenza cytotoxicity displayed by the effector cells. We conclude that the live-attenuated influenza A virus infection induced a broader range of enhanced anti-influenza cytotoxicity than did the inactivated subvirion vaccine.     

Immunity to influenza in ferrets. XIV: Comparative immunity following infection or immunization with live or inactivated vaccine.

Immunization by live influenza virus induced a greater protective effect against subsequent challenge by the homologous virus than by the corresponding killed virus vaccine. Furthermore, tracheas excised from 11-day and 28-day influenza-virus-infected ferrets were more resistant to reinfection than tracheas excised from ferrets immunized by killed influenza vaccine, despite equivalent serum antibody titres at these times. Histological examination of trachea sections taken from vaccinated and virus-infected animals showed an increased cellular inflammatory infiltrate in the latter at Days 11 and 28 after immunization. The amount of IgG detected in these sections, measured by a fluorescent antibody technique, correlated with the extent of cellular infiltration, the fluorescence being both intra- and extracellular for sections from virus-infected animals, but only extracellular in sections from Day-28 vaccinated animals. In contrast there was little or no cellular infiltration into lung tissues, the levels of IgG detected being comparable to those in sections taken from control animals. These results provide further evidence that live influenza vaccines induce local antibody in the upper respiratory tract of ferrets, in contrast to killed influenza vaccines, and that this local induction may play a significant role in the greater protective efficacy of live influenza vaccines.     

Microneedle vaccination with stabilized recombinant influenza virus hemagglutinin induces improved protective immunity

The emergence of the swine-origin 2009 influenza pandemic illustrates the need for improved vaccine production and delivery strategies. Skin-based immunization represents an attractive alternative to traditional hypodermic needle vaccination routes. Microneedles (MNs) can deliver vaccine to the epidermis and dermis, which are rich in antigen-presenting cells (APC) such as Langerhans cells and dermal dendritic cells. Previous studies using coated or dissolvable microneedles emphasized the use of inactivated influenza virus or virus-like particles as skin-based vaccines. However, most currently available influenza vaccines consist of solubilized viral protein antigens. Here we test the hypothesis that a recombinant subunit influenza vaccine can be delivered to the skin by coated microneedles and can induce protective immunity. We found that mice vaccinated via MN delivery with a stabilized recombinant trimeric soluble hemagglutinin (sHA) derived from A/Aichi/2/68 (H3) virus had significantly higher immune responses than did mice vaccinated with unmodified sHA. These mice were fully protected against a lethal challenge with influenza virus. Analysis of postchallenge lung titers showed that MN-immunized mice had completely cleared the virus from their lungs, in contrast to mice given the same vaccine by a standard subcutaneous route. In addition, we observed a higher ratio of antigen-specific Th1 cells in trimeric sHA-vaccinated mice and a greater mucosal antibody response. Our data therefore demonstrate the improved efficacy of a skin-based recombinant subunit influenza vaccine and emphasize the advantage of this route of vaccination for a protein subunit vaccine.     

Production of highly cross-reactive hemagglutination-inhibiting influenza antibodies in ferrets.

Ferrets were sequentially infected at time intervals of 3 weeks with different influenza virus A strains. It was found that secondary infection can result in the appearance of antibodies highly cross-reacting with a virus strain closely related to the strain of first infection. Such highly cross-reacting antibodies were designated as HCR antibodies. Evidence is presented that HCR antibodies were not antineuraminidase antibodies and, in addition, were not specifically oriented to the strain with which the crossing was observed. When using time intervals of 3 weeks between infections, no increase after secondary infection of antibodies oriented to the strain of first infection was recorded. However, when time intervals of 4 to 5 months between infections were used, secondary infections resulted in an increase of antibodies oriented to the strain of first infection ("original antigenic sin") but not in the appearance of HCR antibodies. In addition, antibodies combining specifically with both infecting strains, designated as doubly specific antibodies, were found. Thus, the conclusion was reached that the original antigenic sin phenomenon and the appearance of HCR antibodies are mutually exclusive events.     

Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines

Influenza virus is a globally important respiratory pathogen which causes a high degree of morbidity and mortality annually. The virus is continuously undergoing antigenic change and thus bypasses the host's acquired immunity to influenza. Despite the improvement in antiviral therapy during the last decade, vaccination is still the most effective method of prophylaxis. Vaccination induces a good degree of protection (60-90% efficacy) and is well tolerated by the recipient. For those at risk of complications from influenza, annual vaccination is recommended due to the antigenic changes in circulating strains. However, there is still room for improvement in vaccine efficacy, long-lasting effect, ease of administration and compliance rates. The mucosal tissues of the respiratory tract are the main portal entry of influenza, and the mucosal immune system provides the first line of defence against infection. Secretory immunoglobulin A (SIgA) and IgM are the major neutralizing antibodies directed against mucosal pathogens. These antibodies work to prevent pathogen entry and can function intracellularly to inhibit replication of virus. This review describes influenza virus infection, epidemiology, clinical presentation and immune system response, particularly as it pertains to mucosal immunity and vaccine use. Specifically, this review provides an update of the current status on influenza vaccination and concentrates on the two main types of influenza vaccines currently in use, namely the cold-adapted vaccine (CAV) given intranasally/orally, and the inactivated vaccine (IV) delivered subcutanously or intramuscularly. The commercially available trivalent IV (TIV) elicits good serum antibody responses but induces poorly mucosal IgA antibody and cell-mediated immunity. In contrast, the CAV may elicit a long-lasting, broader immune (humoral and cellular) response, which more closely resembles natural immunity. The immune response induced by these two vaccines will be compared in this review.     

Role of upper respiratory tract infection in the deaths occurring in neonatal ferrets infected with influenza virus

Passive immunization of ferret neonates by colostrally-derived anti-influenza virus IgG did not entirely prevent infection when mothers were immunized with 1 or 2 doses of formalin inactivated vaccine with adjuvant (alhydrogel). Influenza virus replication was almost completely prevented in the lower respiratory tract but only slightly reduced in the upper respiratory tract leading to deaths in about 50% of the neonates. Such neonates showed at most only minor lesions in the lower respiratory tract but moderate to severe inflammatory changes in the upper respiratory tract of most animals. This supports previous results suggesting that deaths, reminiscent of the human sudden infant death syndrome (SIDS), may arise purely as a result of upper respiratory tract infection, possibly following obstruction of the airways.     

Antibody reactive in antibody-dependent cell-mediated cytotoxicity following influenza virus vaccination

The antibody reactive in antibody-dependent, cell-mediated cytotoxicity (ADCC) to influenza virus-infected cells was measured in two groups of seven volunteers each, before and after immunization with inactivated or live attenuated A/Victoria/3/75 influenza virus vaccines. Age-matched controls were seven adult individuals who experienced natural influenza infection due to A/Victoria/3/75-like virus strain. After inactivated whole influenza virus immunization all the subjects showed a significant rise of the antibody reactive in ADCC (from a mean value of 4.7% to 17.1% cytotoxicity, before and 5 weeks after immunization, respectively) as well as of hemagglutination inhibition (HI) antibody (fourfold or greater increase). These immune responses were similar to those observed among naturally infected controls. After live attenuated virus vaccination, no significant increase in titer of antibody reactive in ADCC was detected, even though the vaccine induced significant increase of HI antibody titer. Little correlation was found between ADCC and HI antibody rises in sera of recipients of inactivated virus vaccine and of naturally infected individuals, while, in live attenuated influenza virus vaccinees, the rise of HI antibody titer did not correspond to a significant increase of ADCC antibody titer; several subjects who developed a significant rise in ADCC antibody titer did not show significant variation in antibody to neuraminidase and/or to complement fixation influenza virus antigens.     

Intranasal immunization of ferrets with commercial trivalent influenza vaccines formulated in a nanoemulsion-based adjuvant

NB-1008 is a surfactant-stabilized soybean oil-in-water nanoemulsion (NE) adjuvant with influenza virus antigen incorporated into the NE by simple mixing. Intranasal administration of the antigen with NE adjuvant efficiently produces both mucosal and serum antibody responses as well as a robust cellular Th1 immune response. To demonstrate the adjuvant effect of the W₈₀5EC NE, a killed commercial influenza vaccine for intramuscular administration (Fluzone or Fluvirin) was mixed with the W₈₀5EC NE adjuvant and administered intranasally to naïve ferrets. After a single intranasal immunization, the adjuvanted influenza vaccine elicited elevated serum hemagglutination inhibition (HAI) geometric mean titers (GMTs) ranging from 196 to 905 for the three hemagglutinin (HA) antigens present in the vaccine, which are approximately 19- to 90-fold higher titers at 1/50 the standard intramuscular commercial nonadjuvanted influenza vaccine dose. Seroconversion rates of 67% to 100% were achieved against each of the three viral strains present. The adjuvanted nasal influenza vaccine also produced significant cross immunity to five other H3N2 influenza virus strains not present in the vaccine and produced sterile immunity after challenge with homologous live virus. No safety issues were observed in 249 ferrets receiving the adjuvanted influenza vaccine. These findings demonstrate the ability of W₈₀5EC NE to adjuvant nasally administered influenza vaccine and provide a basis for studying the intranasal W₈₀5EC-adjuvanted influenza vaccine in humans.     

Vesicular stomatitis virus causes abortion and neonatal death in ferrets.

Vesicular stomatitis virus caused transplacental infection in ferrets, resulting in infant death. No clinically significant illness was observed in the adult, despite histological changes in the uterus, liver, and spleen.     

Persistence of cell-mediated immunity to influenza A virus in mice.

Cytotoxic T-cell (Tc) immunity to influenza A virus in mice persists for at least 2 years. Both T-helper (Th) and Tc cells are present and fully functional in old mice; however, the frequency of memory Tc-cell precursors declines with age.     

The immune response of hamsters to purified haemagglutinins and whole influenza virus vaccines following live influenza virus infection

The ability of several, live type A influenza viruses to enhance the serum haemagglutination-inhibiting (HI) antibody response of hamsters to subsequent immunization with inactivated, heterotypic influenza virus vaccines was examined. Live influenza viruses were found to vary in their priming ability for a given vaccine, and a given virus was not able to prime for all inactivated vaccines to an equal extent. Common determinants in the haemagglutinin antigens of the priming virus and the vaccine virus were suggested as responsible for the enhancement of the antibody response to some of the vaccines, but for other pairs of viruses the haemagglutinin antigens were distinct. Thus, enhancement in these instances cannot be due to cross-reacting haemagglutinins. Pre-infection of hamsters by several influenza type A viruses was employed in an attempt to enhance the serum HI antibody response to purified, haemagglutinin antigens prepared from A/PR/8/34 and the MRC-2 recombinant strain of A/England/42/72 viruses. Although prior infection enhanced the antibody response to whole virus, this was not demonstrable for the purified haemagglutinin components of the virus. The possible reasons for this are discussed.     

Oral vaccination of dogs with recombinant rabies virus vaccines

Oral rabies virus (RV) vaccines are used to immunize a diversity of mammalian carnivores, but no single biological is effective for all major species. Recently, advances in reverse genetics have allowed the design of recombinant RV for consideration as new vaccines. The objective of this experiment was to examine the safety, immunogenicity and efficacy of recombinant RV vaccines administered to captive dogs by the oral route, compared to a commercial vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine. Animals consisted of naive purpose-bred beagles of both sexes, and were 6 months of age or older. Dogs were randomly assigned to one of six groups, and received either diluent or vaccine (PBS; V-RG; RV SN10-333; RV SPBN-Cyto c; RV SPBNGA; RV SPBNGAGA), with at least six animals per group. On day 0, 1 ml of each vaccine (or PBS) was administered to the oral cavity of each dog, at an approximate concentration of 10(8) to 10(9) TCID50. After vaccination, dogs were observed daily and bled weekly, for 5 weeks, prior to RV challenge. No signs of illness related to vaccination were detected during the observation period. Excluding the controls, RV neutralizing antibodies were detected in the majority of animals within 1-2 weeks of primary vaccination. Thereafter, all dogs were inoculated in the masseter muscle with a street virus of canine origin. All control animals developed rabies, but no vaccinates succumbed, with the exception of a single dog in the V-RG group. Review of these preliminary data demonstrates the non-inferiority of recombinant RV products, as concerns both safety and efficacy, and supports the suggestion that these vaccines may hold promise for future development as oral immunogens for important carnivore species, such as dogs.     

Influence of prior cellular immunity on the in vitro lymphocyte response to virus antigens after influenza vaccination.

Infections with Vibrio vulnificus resulting in septicemia and high mortality have been correlated with pre-existing liver disease and hemochromatosis. As these conditions are associated with impaired iron metabolism and as iron availability in the host has been implicated in the pathogenicity of a number of bacterial infections, the role of iron as a possible factor in the pathogenesis of V. vulnificus was examined. Injection of mice with iron resulted in a lowering of the 50% lethal dose from 10(6) to 1.1 cells and in a reduction in the time of death postinfection. Elevated serum iron levels were also produced by damaging livers with injections of CCl4. The inoculum size required to kill these mice was directly correlated with serum iron levels. Since the portal of infection of this organism may be ingestion of contaminated seafood, the effects of iron upon orally induced infection were also studied. The effects of adding iron, transferrin, or Desferal (an iron chelate) upon the growth of V. vulnificus in human and rabbit sera were also examined. Iron appeared to be the limiting factor in the ability of this organism to survive or grow in mammalian sera. These results, both in vitro and in vivo, provided strong evidence that iron may play a major role in the pathogenesis of V. vulnificus.