The Safety of Adjuvanted Vaccines Revisited: Vaccine-Induced Narcolepsy.

Despite the very high benefit-to-risk ratio of vaccines, the fear of negative side effects has discouraged many people from getting vaccinated, resulting in the reemergence of previously controlled diseases such as measles, pertussis and diphtheria. This fear has been amplified more recently by multiple epidemiologic studies that confirmed the link of an AS03-adjuvanted pandemic influenza vaccine (Pandemrix, GlaxoSmithKline Biologicals, Germany) used in Europe during the 2009 H1N1 influenza pandemic [A(H1N1) pdm09] with the development of narcolepsy, a chronic sleep disorder, in children and adolescents. However, public misperceptions of what adjuvants are and why they are used in vaccines has created in some individuals a closed "black box" attitude towards all vaccines. The focus of this review article is to revisit this "black box" using the example of narcolepsy associated with the European AS03-adjuvanted pandemic influenza vaccine.

Synthetic peptides non-covalently bound to bacterial hsp 70 elicit peptide-specific T-cell responses in vivo.

We have examined the immunogenicity of complexes formed by non-covalent association of a synthetic peptide corresponding to influenza A virus nucleoprotein, residues 206-229 (pNP) and Mycobacterium tuberculosis heat-shock protein 70 (hsp 70). One or two injections of these complexes given to BALB/c mice without any additional adjuvant, were capable of eliciting very strong peptide-specific proliferative T-cell responses in the spleen. These responses were dependent on the stability of the complex since immunogenicity was lost when dissociated with ATP prior to immunization. T-cell responses to hsp 70 were easily generated by immunization with the purified chaperone alone, either after primary or secondary immunization. Injection of pNP-hsp 70 complexes, however, although generating good primary responses, resulted in very much decreased proliferative responses to the hsp 70.

Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes.

We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the synthesis of several viral proteins. If ultraviolet (UV)-inactivated or bromelain-treated viruses were used, viral protein synthesis could not be detected, and there was poor induction of CTLs. This indicated that dendritic cells were not capable of processing noninfectious virus onto major histocompatibility complex (MHC) class I molecules. However, UV-inactivated and bromelain-treated viruses were presented efficiently to class II-restricted CD4+ T cells. The CD4+ T cells crossreacted with different strains of influenza and markedly amplified CTL formation. Cell lines that lacked MHC class II, and consequently the capacity to stimulate CD4+ T cells, failed to induce CTLs unless helper lymphokines were added. Similarly, dendritic cells pulsed with the MHC class I-restricted nucleoprotein 147-155 peptide were poor stimulators in the absence of exogenous helper factors. We conclude that the function of dendritic cells as APCs for the generation of virus-specific CTLs in vitro depends measurably upon: (a) charging class I molecules with peptides derived from endogenously synthesized viral antigens, and (b) stimulating a strong CD4+ helper T cell response.

Induction of influenza A virus cross-reactive cytotoxic T cells by a nucleoprotein/haemagglutinin preparation.

An ammonium deoxycholate fraction from bromelain-treated influenza A virus was highly enriched for virus nucleoprotein and contained residual haemagglutinin (NP/HA). The preparation did not contain detectable levels of matrix or neuraminidase proteins and was free of infectious virus. NP/HA effectively primed mice for cytotoxic T cells which lysed syngeneic cells infected with any type A influenza virus. Furthermore, NP/HA generated A-type virus cross-reactive cytotoxic T cells when added in vitro to spleen cells from mice previously primed with infectious influenza A virus. These properties imply that NP/HA has potential as a vaccine for heterotypic influenza A immunity.

Hybridoma antibodies produced against bromelain derived cores of influenza virus.

Splenic lymphocytes from BALB/c mice immunized with "cores" of influenza virus, obtained after bromelain cleavage of the surface glycoprotein, were fused with the P3-NS1/1-Ag-1 mouse cell line to yield hybridoma cultures. Among 20 stable cloned hybrid cells secreting monoclonal antibodies, one was specific for the nucleoprotein (NP), 11 were specific for the membrane (M) protein and eight were specific for the hemagglutinin (HA). These "cores" used as immunogen contained only the internal proteins of the influenza virus, namely the three polymerases, the NP and the M protein and no HA when examined by standard procedures of SDS-PAGE, electron microscopy and hemagglutination activity. It thus appeared that a small amount of contaminating antigens can sensitize a sufficient number of mouse B cells to be selected as hybrid partners. These antibodies were provisionally assigned as anti-carbohydrate attached to the HA.

Blocking of influenza virus-induced cell-mediated cytotoxicity by hemagglutinin-specific monoclonal antibody.

The relative importance of three major proteins of influenza virus in the mechanism of induction of cell-mediated cytotoxicity (natural killer cell activity) was assessed by an overnight chromium-51-release assay using radiolabeled K-562 cells as target cells and Ficoll-Hypaque-purified peripheral-blood lymphocytes as effector cells. Incubation of peripheral-blood lymphocytes with influenza virus (whether type-A or type-B) showed that intact and formalin-inactivated influenza virus enhanced cell-mediated cytotoxicity equally. The stimulation by intact or inactivated virions was comparable to that induced by the two major internal mediators of positive natural killer cell regulation, namely interferon and interleukin-2. This virus-induced cell-mediated cytotoxicity, which was mediated by human natural-killer 1+ cells, could be blocked only with monoclonal antibodies to the hemagglutinin and not with antinucleoprotein or antimatrix protein monoclonal antibodies, results indicating that the hemagglutinin of influenza virus is a potent mediator of natural killer cell stimulation in vitro.