Protection by humoral elements independent of virus neutralization activity against an influenza virus challenge.

To investigate the protective efficacy of a seasonal trivalent inactivated whole virion influenza vaccine (TIV) adjuvanted with aluminum phosphate (Fluval AB, referred to here as TIV+Al), we immunized mice with the TIV+Al, and as controls, with TIV, TIV+Al combined with Freund adjuvant (TIV+Al+F), inactivated A/PR/8/34(H1N1) (PR8) strain or PBS, and challenged them with a lethal dose of a mouse-adapted PR8 virus. Serum pools from immunized mice were passively transferred to recipient mice that were then challenged similarly. All actively immunized mice survived the challenge. Of recipient mice receiving serum from mice actively immunized with TIV, TIV+Al or TIV+Al+F, 20%, 80%, and 100% survived, respectively. Rates of mortality and morbidity of recipient mice were inversely proportional to the hemagglutination inhibition (HI) antibody level to the vaccine virus in the absence of detectable PR8-specific HI, neuraminidase inhibition (NI) and virus neutralization (VN) antibodies. No cross-reactivity was observed between vaccine and PR8 strains in in vitro HI, NI or VN assays. In splenocytes from TIV+Al-immunized mice production of IFN-γ or granzyme-B protein and mRNA expression increased (p<0.05). Thus, antibodies play a major role in the protection against a mismatched challenge infection independent of HI, NI and VN activity, but cellular immune responses may contribute to full protection in actively immunized mice.

Protection of Chinese painted quails (Coturnix chinensis) against a highly pathogenic H5N1 avian influenza virus strain after vaccination.

Chinese painted quails immunized with a single dose (6 µg HA) of inactivated H5N1 (clade 1) influenza vaccine NIBRG-14 and challenged with 100 LD50 of the heterologous A/Swan/Nagybaracska/01/06(H5N1) (clade 2.2) strain were protected, whereas unvaccinated quails died after challenge. No viral antigens or RNA were detected in cloacal swabs from immunized animals. Sera obtained post-immunization gave low titres in serological assays against the vaccine and the challenge viruses. Our results demonstrate the protective efficacy of the NIBRG-14 strain against the challenge virus and the usefulness of these small birds in protection studies of influenza vaccines.

[Importance of the case of coronavirus-associated severe acute respiratory syndrome detected in Hungary in 2005]. / Miért aktuális 2013-ban a súlyos akut respirációs szindrómát okozó coronavírus-fertozés 2005-ben Magyarországon igazolt esete?

Ten years have elapsed since the severe acute respiratory syndrome outbreak, which resulted in more than 8000 cases worldwide with more than 700 deaths. Recently, a new coronavirus, the Middle East Respiratory Syndrome Coronavirus emerged, causing serious respiratory cases and death. By the end of August 2013, 108 cases including 50 deaths were reported. The authors discuss a coronavirus-associated severe acute respiratory syndrome, which was detected in Hungary in 2005 and highlight its significance in 2013. In 2005 the patient was hospitalized and all relevant clinical and microbiological tests were performed. Based on the IgG antibody positivity of the serum samples, the patient was diagnosed as having severe acute respiratory syndrome coronavirus infection in the past. The time and source of the infection remained unknown. The condition of the patient improved and he was discharged from the hospital. The case raises the possibility of infections in Hungary imported from remote areas of the world and the importance of thorough examination of patients with severe respiratory syndrome with unknown etiology.

High-level cellular and humoral immune responses in Guinea pigs immunized intradermally with a heat-inactivated varicella-zoster virus vaccine.

The threat of varicella and herpes zoster in immunocompromised individuals necessitates the development of a safe and effective varicella-zoster virus (VZV) vaccine. The immune responses of guinea pigs to the intradermal (i.d.) or subcutaneous (s.c.) administration of a heat-inactivated or live VZV vaccine were investigated. Relative to nonimmunized animals, a single 399-PFU dose of vaccine induced nonsignificant increases in gamma interferon (IFN-γ), granzyme B, and perforin mRNA expression in the splenocytes of all groups, while two i.d. administrations of the inactivated vaccine increased IFN-γ mRNA expression significantly (P < 0.005). A single 1,995-PFU dose significantly increased the expression of IFN-γ mRNA in the groups receiving the vaccine either i.d. (P < 0.005) or s.c. (P < 0.05), that of granzyme B mRNA in the groups immunized i.d. with the inactivated (P < 0.005) or live (P < 0.005) vaccine, and that of perforin mRNA in the animals that received the inactivated vaccine i.d. (P < 0.005). Importantly, increases in the expression of IFN-γ (P = 0.025), granzyme B (P = 0.004), and perforin (P > 0.05) mRNAs were observed in the animals immunized i.d. with 1,995 PFU of inactivated vaccine relative to those immunized s.c. with the same dose. The proportion of animals expressing IFN-γ mRNA mirrored the proportion expressing IFN-γ protein (correlation coefficient of 0.88). VZV glycoprotein-specific and virus-neutralizing antibodies were produced with no significant intergroup differences. A booster i.d. administration of the 399-PFU dose of heat-inactivated vaccine enhanced the antibody responses. These results demonstrate that i.d. administration of an inactivated VZV vaccine can be an efficient mode of immunization against VZV.

Standardization and validation of assays determining cellular immune responses against influenza.

Influenza vaccine efficacy does not always correlate with humoral immune responses. Recent reports indicate that the cellular immune response also contributes to protection, however robust assays are lacking. We standardized and validated assays for detection of human influenza-specific cellular responses in four international laboratories. The production of granzyme B as marker of T cell-mediated cytotoxicity and release of Th1 and Th2 cytokines were evaluated. The granzyme B and cytokine assays were specific, accurate, precise, and robust. Replicate stimulations with PBMC from the same donors showed an intra-laboratory robustness (coefficient of variation) for quantitation of granzyme B of 33% and for cytokines - including IFN-gamma, TNF-alpha, IL-2, IL-10, IL-4, IL-13, GM-CSF and including the log IFN-gamma/IL-10 ratio - of 52%. The inter-laboratory robustness for detection of granzyme B was 29% and for detection of all cytokines was 49%. The assays can now be used for determining cell-mediated immunity and explored as correlates of protection. Moreover, the precision and robustness of these cellular assays allow the reliable detection of cellular responses even in small study populations.

Yearly licensing studies from 1997 to 2007 of the inactivated whole virus seasonal influenza vaccine fluval--a useful approach to pandemic vaccine development even in less well developed countries?

OBJECTIVE: Seasonal vaccination has been consistently shown to significantly reduce morbidity and mortality because of influenza epidemics, even in healthy, working adults. Here we report the results of the yearly licensing studies of the past 11 influenza seasons (1997-2007) with a trivalent, inactivated whole virus vaccine with an aluminum phosphate adjuvant system. METHODS: Sixty healthy volunteers per age group (18-60 years and 60 years and older) were enrolled to receive vaccination each year, thus, a total of 1080 subjects were studied. Serum antibody titers were measured by hemagglutination inhibition (HI). RESULTS: The vaccine met the criteria for licensing each year, meaning seroprotection (achievement of an HI titer of >1:40 in >70% of subjects); seroconversion, i.e. a >4-fold increase in HI antibody titer, or reaching a titer of >1:40, in >40% of subjects; and an increase in geometric mean titers by >2.5-fold. Side effects were rare and mild. The same method was used to produce a pre-pandemic vaccine against influenza A (H5N1), which has been shown to be safe and immunogenic in humans. CONCLUSIONS: We conclude that the method presented is safe, effective and may serve as a useful approach to seasonal and pandemic vaccine production even in less well-developed countries by means of technological transfer.