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1.  Innate Responses Induced by Whole Inactivated Virus or Subunit Influenza Vaccines in Cultured Dendritic Cells Correlate with Immune Responses In Vivo 
PLoS ONE  2015;10(5):e0125228.
Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates.
PMCID: PMC4416761  PMID: 25933037
2.  Superior Immunogenicity of Inactivated Whole Virus H5N1 Influenza Vaccine is Primarily Controlled by Toll-like Receptor Signalling 
PLoS Pathogens  2008;4(8):e1000138.
In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.
Author Summary
The rise and spread of the highly pathogenic avian H5N1 influenza virus has seriously increased the risk of a new influenza pandemic. However, the number of vaccine doses that can be produced with today's production capacity will fall short of the demand in times of a pandemic. Use of inactivated whole virus (WIV) vaccines, which are more immunogenic than split virus or subunit vaccines in an unprimed population, could contribute to a dose-sparing strategy. Yet, the mechanisms underlying the superior immunogenicity of WIV vaccine formulations are unknown. Here, we demonstrate that the viral RNA present in inactivated virus particles is crucial for the improved immunogenic properties of WIV in mice. By triggering Toll-like receptor 7 (TLR7), the viral RNA activates innate immune mechanisms that augment and determine subsequent adaptive responses. Efficient TLR7 signalling is lost in split virus and subunit vaccines with the processing steps that lead to disruption of the integrity of the virus particle and exclusion of the RNA. Our results prove for the first time to our knowledge that the immune-potentiating mechanism of a classic vaccine is based on activation of the innate immune system by one of its structural components. These findings may reflect a general principle for viral vaccines and provide a rational basis for further improvement of influenza vaccines, which are urgently needed in the face of the current H5N1 pandemic threat.
PMCID: PMC2516931  PMID: 18769719
3.  Cryoprecipitate in the Treatment of Hemophilia 
California Medicine  1970;113(2):66-67.
PMCID: PMC1501487  PMID: 18730395

Results 1-3 (3)