Nella J. Nieuwkoop

Antigenic Drift in the Influenza A Virus (H3N2) Nucleoprotein and Escape from Recognition by Cytotoxic T Lymphocytes

ABSTRACT Viruses exploit different strategies to escape immune surveillance, including the introduction of mutations in cytotoxic T-lymphocyte (CTL) epitopes. The sequence of these epitopes is critical for their binding to major histocompatibility complex (MHC) class I molecules and recognition by specific CTLs, both of which interactions may be lost by mutation. Sequence analysis of the nucleoprotein gene of influenza A viruses (H3N2) isolated in The Netherlands from 1989 to 1999 revealed two independent amino acid mutations at the anchor residue of the HLA-B27-specific CTL epitope SRYWAIRTR (383 to 391). A R384K mutation was found in influenza A viruses isolated during the influenza season 1989–1990 but not in subsequent seasons. In the influenza season 1993–1994, a novel mutation in the same CTL epitope at the same position was introduced. This R384G mutation proved to be conserved in all influenza A viruses isolated from 1993 onwards. Both mutations R384K and R384G abrogated MHC class I presentation and allowed escape from recognition by specific CTLs.

A randomized, double blind study in young healthy adults comparing cell mediated and humoral immune responses induced by influenza ISCOM vaccines and conventional vaccines

Although current influenza vaccines have been shown to reduce influenza-related morbidity and mortality, there is a desire to develop more efficacious products. Vaccines which can induce CD8(+) cytotoxic T cell (CTL) responses in addition to strong antibody responses may be more effective in preventing disease since it has been demonstrated that CTL contribute to protective immunity, even against drift variants of influenza A viruses. The immunogenicity of two types of experimental influenza vaccines, which were based on immune stimulating complexes (ISCOM), were evaluated and compared with a conventional non-adjuvanted inactivated split virion vaccine, after immunization of human volunteers. In this randomized, double blind study, it was shown that the ISCOM vaccines altered the kinetics of the serum antibody response, resulting in more rapid titer rises against the vaccine strains. This accelerated antibody response coincided with enhanced in vitro proliferative T cell responses, which were observed shortly after vaccination. In addition, CTL responses were observed in a higher proportion of the vaccinees receiving an ISCOM vaccine, than in vaccinees receiving the conventional influenza vaccine.

Cross-protective immunity against influenza pH1N1 2009 viruses induced by seasonal influenza A (H3N2) virus is mediated by virus-specific T-cells.

Influenza A (H1N1) viruses of swine origin were introduced into the human population in 2009 and caused a pandemic. The disease burden in the elderly was relatively low, which was attributed to the presence of cross-reacting serum antibodies in this age group, which were raised against seasonal influenza A (H1N1) viruses that circulated before 1957. It has also been described how infection with heterosubtypic influenza viruses can induce some degree of protection against infection by a novel strain of influenza virus. Here, we assess the extent of protective immunity against infection with the 2009 influenza A (H1N1) pandemic influenza virus that is afforded by infection with a seasonal influenza A (H3N2) virus in mice. Mice that experienced a primary A (H3N2) influenza virus infection displayed reduced weight loss after challenge infection and cleared the 2009 influenza A (H1N1) virus infection more rapidly. To elucidate the correlates of protection of this heterosubtypic immunity to pandemic H1N1 virus infection, adoptive transfer experiments were carried out by using selected post-infection lymphocyte populations. Virus-specific CD8(+) T-cells in concert with CD4(+) T-cells were responsible for the observed protection. These findings may not only provide an explanation for epidemiological differences in the incidence of severe pandemic H1N1 infections, they also indicate that the induction of cross-reactive virus-specific CD8(+) and CD4(+) T-cell responses may be a suitable approach for the development of universal influenza vaccines.

A mutation in the HLA-B*2705-restricted NP383-391 epitope affects the human influenza A virus-specific cytotoxic T-lymphocyte response in vitro.

ABSTRACT Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in or adjacent to CTL epitopes. Recently, an amino acid substitution (R384G) in an HLA-B*2705-restricted CTL epitope in the influenza A virus nucleoprotein (nucleoprotein containing residues 383 to 391 [NP383-391]; SRYWAIRTR, where R is the residue that was mutated) was associated with escape from CTL-mediated immunity. The effect of this mutation on the in vitro influenza A virus-specific CTL response was studied. To this end, two influenza A viruses, one with and one without the NP383-391 epitope, were constructed by reverse genetics and designated influenza viruses A/NL/94-384R and A/NL/94-384G, respectively. The absence of the HLA-B*2705-restricted CTL epitope in influenza virus A/NL/94-384G was confirmed by using 51Cr release assays with a T-cell clone specific for the NP383-391 epitope. In addition, peripheral blood mononuclear cells (PBMC) stimulated with influenza virus A/NL/94-384G failed to recognize HLA-B*2705-positive target cells pulsed with the original NP383-391 peptide. The proportion of virus-specific CD8+ gamma interferon (IFN-γ)-positive T cells in in vitro-stimulated PBMC was determined by intracellular IFN-γ staining after restimulation with virus-infected autologous B-lymphoblastoid cell lines and C1R cell lines expressing only HLA-B*2705. The proportion of virus-specific CD8+ T cells was lower in PBMC stimulated in vitro with influenza virus A/NL/94-384G obtained from several HLA-B*2705-positive donors than in PBMC stimulated with influenza virus A/NL/94-384R. This finding indicated that amino acid variations in CTL epitopes can affect the virus-specific CTL response and that the NP383-391 epitope is the most important HLA-B*2705-restricted epitope in the nucleoprotein of influenza A viruses.

Functional Compensation of a Detrimental Amino Acid Substitution in a Cytotoxic-T-Lymphocyte Epitope of Influenza A Viruses by Comutations

ABSTRACT Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs.

Introduction of the haemagglutinin transmembrane region in the influenza virus matrix protein facilitates its incorporation into iscom and activation of specific CD8+ cytotoxic T lymphocytes.

The gene encoding the influenza virus A matrix (MA) protein was cloned into the bacterial expression vector pMalC with and without the sequence encoding the transmembrane region of the haemagglutinin (HA). With the resulting recombinant proteins, immune stimulating complexes (ISCOM) were prepared. The MA protein with the hydrophobic anchor region (rMAHA) associated more efficiently with ISCOM than the unmodified MA protein (rMA). A B-lymphoblastoid cell line (B-LCL) was lysed by an autologous CD8(+) cytotoxic T lymphocyte (CTL) clone specific for the MA protein after incubation with rMAHA-ISCOM but not after incubation with rMA, rMAHA, rMA-ISCOM or empty ISCOM. The B-LCL was also lysed by the CTL clone after incubation with empty ISCOM mixed with the respective MA proteins. Incubation of ISCOM with the rMAHA protein proved to be the most efficient in this respect. Addition of the proteasome inhibitors lactacystin or clasto-lactacystin beta-lactone to the B-LCL incubated with rMAHA-ISCOM or the MA proteins mixed with empty ISCOM dramatically decreased the lysis by the CD8(+) CTL clone. These results indicate that the addition of a hydrophobic anchor to hydrophilic proteins in combination with ISCOM facilitates their entry in the MHC class I processing and presentation pathway. This may be an attractive approach for the development of subunit vaccines aiming at the induction of CTL-mediated immunity.

Antigen processing for MHC class I restricted presentation of exogenous influenza A virus nucleoprotein by B-lymphoblastoid cells.

In general, exogenous proteins are processed by antigen‐presenting cells in the endosomes for major histocompatibility complex (MHC) class II presentation to CD4+ T cells, while proteins synthesized endogenously are processed in the cytoplasm for MHC class I presentation to CD8+ T cells. However, it is recognized that exogenous proteins can be processed for MHC class I presentation also, and evidence in favour of alternatives to the conventional MHC class I processing and presentation pathway is accumulating. Here, we show that exogenous recombinant influenza A virus nucleoprotein (rNP) is processed for MHC class I presentation to CD8+ cytotoxic T lymphocytes (CTL) by EBV‐transformed, B‐lymphoblastoid cell lines (B‐LCL). Processing of rNP for HLA‐B27‐associated presentation seemed to follow the conventional MHC class I pathway predominantly, as presentation was diminished in the presence of lactacystin and brefeldin A, but was less sensitive to chloroquine and NH4Cl. HLA‐B27‐associated presentation was also observed using cells lacking a functional transporter associated with antigen processing, suggesting that alternative pathways may be exploited for processing of rNP.

Binding of DC-SIGN to the Hemagglutinin of Influenza A Viruses Supports Virus Replication in DC-SIGN Expressing Cells

Dendritic cells express lectins receptors, like DC-SIGN, which allow these cells to sense glycans that are present on various bacterial and viral pathogens. Interaction of DC-SIGN with carbohydrate moieties induces maturation of dendritic cells and promotes endocytosis of pathogens which is an important property of these professional antigen presenting cells. Uptake of pathogens by dendritic cells may lead to cross-presentation of antigens or infection of these cells, which ultimately results in activation of virus-specific T cells in draining lymph nodes. Little is known about the interaction of DC-SIGN with influenza A viruses. Here we show that a virus with a non-functional receptor binding site in its hemagglutinin, can replicate in cells expressing DC-SIGN. Also in the absence of sialic acids, which is the receptor for influenza A viruses, these viruses replicate in DC-SIGN expressing cells including human dendritic cells. Furthermore, the efficiency of DC-SIGN mediated infection is dependent on the extent of glycosylation of the viral hemagglutinin.

Infection of the upper respiratory tract with seasonal influenza A(H3N2) virus induces protective immunity in ferrets against infection with A(H1N1)pdm09 virus after intranasal, but not intratracheal, inoculation

ABSTRACT The clinical symptoms caused by infection with influenza A virus vary widely and depend on the strain causing the infection, the dose and route of inoculation, and the presence of preexisting immunity. In most cases, seasonal influenza A viruses cause relatively mild upper respiratory tract disease, while sometimes patients develop an acute severe pneumonia. Heterosubtypic immunity induced by previous infections with influenza A viruses may dampen the development of clinical symptoms caused by infection with influenza A viruses of another subtype, as is the case during influenza pandemics. Here we show that ferrets acquire protective immunity after infection of the upper respiratory tract with a seasonal influenza A(H3N2) virus against subsequent infection with influenza A(H1N1)pdm09 virus inoculated by the intranasal route. However, protective heterosubtypic immunity was afforded locally, since the prior infection with the A(H3N2) virus did not provide protection against the development of pneumonia induced after intratracheal inoculation with the A(H1N1)pdm09 virus. Interestingly, some of these animals developed more severe disease than that observed in naïve control animals. These findings are of interest in light of the development of so-called universal influenza vaccines that aim at the induction of cross-reactive T cell responses.

Use of GFP-expressing influenza viruses for the detection of influenza virus A/H5N1 neutralizing antibodies.

The hemagglutination inhibition (HI) assay is used most commonly for the detection of antibodies to influenza viruses. However, for the detection of antibodies to avian influenza viruses of the H5N1 subtype either induced by infection or by vaccination, the HI assay is insensitive. Therefore, the virus neutralization (VN) assay has become the method of choice to detect human serum antibodies directed to these viruses. However, this assay requires a second assay for the detection of residual virus replication, which makes it laborious to perform and less suitable for high throughput testing of large numbers of samples. Here we describe an alternative method for the detection of these antibodies, which is based on the use of reporter viruses that express the green fluorescent protein (GFP) upon infection of target cells. GFP-expressing viruses were generated carrying the HA of a variety of antigenically distinct H5N1 influenza viruses. The method proved easy to perform and could be carried out rapidly. Using a panel of antisera raised against H5N1 influenza viruses, the assay based on GFP expressing viruses was compared with the classical virus neutralization assay and the hemagglutination inhibition assay. In general, the results obtained in these assays correlated well. It was concluded that the assay based on the reporter viruses is an attractive alternative for the classical virus neutralization assay and suitable for large sero-epidemiological studies or for the assessment of vaccine efficacy in clinical trials.