Department of Virology and Molecular Biology1 and Department of Immunology2, St Jude Childrens Research Hospital, Memphis, TN 38105-2794, USA
Author for correspondence: Robert Webster. Fax +1 901 523 2622. e-mail robert.webster{at}stjude.org
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Abstract |
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Introduction |
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It has been established that major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T-lymphocytes (CTLs) play a central role in the clearance of primary influenza virus infections (Doherty et al., 1992 ; Eichelberger et al., 1991
; Epstein et al., 1998
; Graham & Braciale, 1997
). In mouse models, effector CTLs are first detectable in the lung on day 7 and their numbers peak by day 9 or 10 (Allan et al., 1990
; Eichelberger et al., 1991
; Flynn et al., 1998
; Hou & Doherty, 1995
). The accumulation of CTLs correlates with clearance of the virus, which occurs by day 8 or 9, and this clearance depends on either Fas or perforin mechanisms (Kagi & Hengartner, 1996
; Topham et al., 1997
). Antibody is generated late in the primary response and does not play a significant role in clearing primary infection unless the viral dose is high (Graham & Braciale, 1997
; Zhong et al., 2000
). However, pre-existing neutralizing antibodies are completely protective against secondary challenge with the same virus; this protection is the basis of current influenza vaccines. In general, the presence of virus-specific neutralizing antibodies prevents replication of the virus in the lung and blocks the development of symptoms.
Although humoral immunity provides complete protection against secondary challenge with the same virus, it is ineffective against serologically distinct viruses (Ada & Jones, 1986 ; Couch & Kasel, 1983
; Gorman et al., 1992
). In contrast, cellular responses to cross-reactive epitopes (often from internal viral proteins) provide a substantial degree of protection against serologically distinct viruses (Rimmelzwaan & Osterhaus, 1995
; Yewdell et al., 1985
). This form of immunity, referred to as heterosubtypic immunity, is unable to prevent reinfection per se, but can reduce the maximal viral load, mediate faster viral clearance, and provide in animal models a substantial degree of protection against challenge with a lethal dose of virus (Anker et al., 1978
; Flynn et al., 1998
; Liang et al., 1994
; Nguyen et al., 1999
; Rimmelzwaan & Osterhaus, 1995
; Schulman & Kilbourne, 1965
; Schulman et al., 1977
). Mouse studies indicate that heterosubtypic immunity is mediated by both CD4+ and CD8+ T cells, although the CD8+ subset is generally considered to be more important (Liang et al., 1994
; Yap & Ada, 1978
). These T cells are primed during the primary response to infection and then persist in the animal after viral clearance and are able to respond more vigorously to a secondary challenge. Although heterosubtypic immunity has been shown to be capable of controlling secondary influenza virus infections, its effectiveness against highly virulent strains of influenza has not been clearly determined.
The A/HK/156/97 (HK156) virus may have resulted from genetic reassortment between cocirculating H5N1 and H9N2 viruses (Guan et al., 1999 ). One of the H9N2 viruses isolated in the 1997 surveillance of the Hong Kong markets, A/Quail/HK/G1/97 (herein called QHKG1), contains internal genes (i.e. genes that encode internal proteins) that are 98% homologous to those of the HK156 virus. This feature makes QHKG1 a likely candidate with which to study heterosubtypic immunization against HK156. In this report, we show that C57BL/6 mice are protected against a lethal HK156 infection by heterologous immunization with QHKG1. This protection is also observed in µMT knock-out mice that are B-cell-deficient and therefore are incapable of producing an antibody response. These results have important implications for the development of novel strategies against HK156 infection and future influenza vaccine candidates.
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Methods |
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C57BL/6 and BALB/c mice were purchased from Jackson Laboratory and the B-cell-deficient µMT mice (Kitamura et al., 1991 ) were bred in the Animal Resource Center at St Jude Childrens Research Hospital (Riberdy et al., 1999
). All mice were maintained in specific-pathogen-free conditions prior to infection.
All immunizations were done with live preparations of virus. Briefly, mice were immunized with 4·5 log10 egg infectious doses (EID50) of QHKG1 and challenged with 2·8 log10 EID50 of HK156 or 4·2 log10 EID50 of R103 (a virus generated in our laboratory by reassortment of GSHK437 and HK1073). All viruses were administered intranasally to mice anaesthetized with Avertin (2,2,2-tribromoethanol). Infection with QHKG1 typically results in loss of up to 15% of the initial weight, but the mice recover and become healthy again. Additional pathological effects are not observed in these mice. Secondary challenge was done 4 weeks after primary infection, and the survival of the mice was monitored daily.
Generation of reassortant virus.
MadinDarby canine kidney (MDCK) cells were used to generate reassortant viruses. Briefly, the GSHK437 and HK1073 viruses were mixed and incubated for 30 min at 4 °C before infection at 37 °C for 30 min. The cells were washed and incubated overnight at 37 °C. The next day, supernatant was harvested and neutralized with antiserum to H9N2. Virus from the neutralized supernatant was plaque purified twice before it was produced on a large scale. The pathogenic potential of the candidate reassorted viruses was tested by infecting BALB/c mice (4·2 log10 EID50). In addition, haemagglutinin and neuraminidase inhibition assays were performed as previously described (Palmer et al., 1975 ). To sequence the viral genomes, RNA was obtained with the RNeasy Kit (Qiagen) and reverse transcribed by using AMV reverse transcriptase (Life Sciences). Amplification of cDNA was then accomplished by using TaKaRa Ex Taq (Panvera). Sequencing reactions were performed by the Center for Biotechnology at St Jude Childrens Research Hospital. Template DNA was sequenced by using rhodamine or dRhodamine dye-terminator cycle sequencing ready reaction kits with AmpliTaq DNA polymerase FS (Perkin-Elmer, Applied Biosystems), and synthetic oligonucleotides. The samples were analysed on PE/ABI model 373, model 373 Stretch or model 377 DNA sequencers (Perkin-Elmer, Applied Biosystems).
RMA-S stabilization assay.
To determine MHC restriction, peptide binding to Kb or Db molecules in RMA-S cells was performed as previously described (Cole et al., 1997 ). These cells internalize MHC molecules very rapidly unless they are bound to a peptide, in which case they become stabilized in the cell membrane. Briefly, RMA-S cells were grown at 31 °C overnight at a density of 5x105 cells/ml. The next day, 105 cells were seeded per well of a 96-well plate containing different concentrations of the peptide to be analysed. The cells were incubated at 25 °C for 30 min and then at 37 °C for 3 h. After the cells had been stained with monoclonal antibodies to Kb or Db, cell surface stabilization was analysed by flow cytometry using FACScan and Cell Quest software (Becton Dickinson). The peptides used in this assay were A/PuertoRico/8/34 (PR8) NP366374 (ASNENMETM), HK156 VA366374 (ASNENVEAM), vesicular stomatitis virus NP5259 (RGYVYQGL) and Sendai virus NP324332 (FAPGNYPAL). All peptides were synthesized in a PE/ABI 433 Peptide Synthesizer (Perkin-Elmer, Applied Biosystems) at the Center for Biotechnology, St Jude Childrens Research Hospital.
Cytotoxic assays.
Cytotoxic activity was analysed by using the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega). This assay measures the release of endogenous lactate dehydrogenase enzyme instead of radioactive chromium. Bronchoalveolar lavage (BAL) was performed 10 days after infection, and adherent cells in the specimens were removed by allowing them to adhere to plastic. The percent cytotoxicity was calculated by using the following formula: % cytotoxicity =[(experimental-spontaneous)/(maximal-spontaneous)]x100.
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Results |
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Role of cellular immunity in the protection against HK156
To formally rule out a role for humoral immunity in protection against HK156 infection after QHKG1 immunization we took advantage of B-cell-deficient mice (µMT), which are unable to make an antibody response (Kitamura et al., 1991 ; Riberdy et al., 1999
). Naïve µMT mice succumbed to HK156 infection in a manner similar to that of wild-type C57BL/6 mice inasmuch as 100% of the mice were dead by day 9. µMT mice that had been previously immunized with QHKG1 were partially protected against HK156 challenge inasmuch as there was a significant delay in the death of most of the mice (Table 4
). For example, 57% of the QHKG1-immunized µMT mice were still alive at day 9, a timepoint when all of the naïve µMT mice had died. In addition, some of the QHKG1-immunized µMT mice survived until day 10. A log-rank test for difference in survival between both groups revealed that there is evidence to suggest that the QHKG1-immunized mice had better survival than the naïve group. The observation that µMT mice previously immunized with QHKG1 finally succumbed to HK156 challenge was not surprising since these mice are more susceptible to influenza virus infection than wild-type C57BL/6 mice (Graham & Braciale, 1997
; Riberdy et al., 1999
). However, these data confirm that previous immunization with QHKG1 induces heterosubtypic immunity against the HK156 virus.
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Discussion |
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It is generally believed that cellular immune responses have limited capacity to protect animals from a lethal dose of virus. Although a recall T cell response to infection is much faster than a primary response, it nevertheless takes several days to develop, during which time the virus can multiply to high titre. Previous studies have shown that mice can be protected from a lethal challenge with PR8 after priming with x31, a laboratory reassortant that differs from PR8 (H1N1) only in its envelope proteins (H3N2). However, this protection is relatively limited (it does not protect against very high doses of virus) and tends to wane rapidly (Liang et al., 1994 ). In this regard, it might have been anticipated that heterosubtypic immunity would be ineffective against challenge with a highly virulent virus such as HK156. Therefore, our results are significant since they provide the first demonstration that protective cell-mediated immunity can be established against the highly virulent HK156 virus.
The mechanism of heterosubtypic immunity is poorly understood. CD8+ T cells appear to play a major role in controlling virus replication (Eichelberger et al., 1991 ). However, it is also apparent that CD4+ cells can also be important (Zhong et al., 2000
). Moreover, CD4+ T cells seem to be more important in the immune response after DNA vaccination (Epstein et al., 2000
; Ulmer et al., 1998
). In the current studies we have not distinguished whether the protective immunity was mediated by either CD4+ or CD8+ T cells due to the difficulties of working with the HK156 virus. However, we did demonstrate that QHKG1 induced a CTL response and postulate that this is a major contributor to the heterosubtypic immunity observed. Very little information regarding cellular immunity against more pathogenic strains such as H5N1 has been reported. To our knowledge, only one group has reported data regarding a cellular response against HK156 and it was an indirect measure of cross-protection in humans that have been previously infected with circulating influenza virus strains (Jameson et al., 1999
). In the current studies we were able to determine that heterosubtypic immunity could be generated when only two viral genes were homologous. The fact that this protection can be established despite only two homologous proteins being shared by the viruses used warrants further investigation.
In the course of these studies we generated an H5N1 reassortant that contains six genes from the GSHK437 (H5N1) and two genes (NP and PB2) from the HK1073 (H9N2) non-pathogenic parents. The reassortant is highly pathogenic and implicates NP and PB2 as having an important contribution to pathogenesis when present with the highly cleavable H5 of the GSHK437 isolate. The presence of the highly cleavable H5 is not sufficient to confer high pathogenicity, as the GSHK437 H5N1 virus is not pathogenic. Similarly, the presence of the HK156-like NP and PB2 genes in the absence of the H5 (as in the case of the HK1073 virus) does not result in virulence (Fig. 2). A likely explanation for this observation could be that the combination of NP, PB2 and HA genes from HK156 provides increased replication ability relative to the parental viruses. This point remains to be elucidated. With the advent of new and more efficient methods like the recently described reverse genetics system (Neumann et al., 1999
), we should be able to generate additional reassortants to further characterize the contribution of each of the HK156-like genes to pathogenesis and the impact of having each one of those genes on the cellular response of the infected host. Nevertheless, this study provides findings that should contribute to the understanding of the pathogenicity of H5N1 influenza viruses.
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Acknowledgments |
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This work was supported by National Institute of Health grants AI07372 (E.O.), AI95357 (R.G.W.) and AI37597 (D.L.W.); by the Cancer Center Support CORE grant P30 CA21765; by the American Lebanese Syrian Associated Charities (ALSAC); and by the Trudeau Institute.
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Footnotes |
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References |
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Received 9 June 2000;
accepted 2 August 2000.