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  • Antibody Response to Flu Virus Modifies CD8 T cell responses

    An unexpected antibody response to an engineered influenza virus modifies CD8<SUP>+</SUP> T cell responses
    </NOBR><NOBR>Paul G. Thomas<SUP>*</SUP></NOBR>, <NOBR>Scott A. Brown<SUP>*</SUP></NOBR>, <NOBR>Wen Yue<SUP>*</SUP></NOBR>, <NOBR>Jenny So<SUP>*</SUP></NOBR>, <NOBR>Richard J. Webby<SUP></SUP></NOBR>, and <NOBR>Peter C. Doherty<SUP>*</SUP><SUP>,</SUP></NOBR>
    Departments of *Immunology and <SUP></SUP>Infectious Diseases, St. Jude Children?s Research Hospital, Memphis, TN 38105

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    <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> Abstract </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
    Abstract
    Results
    Discussion
    Materials and Methods
    Acknowledgements
    References
    </TH></TR></TBODY></TABLE>

    The ovalbumin<SUB>323?339</SUB> peptide that binds H2I-A<SUP>b</SUP> was engineered<SUP> </SUP>into the globular heads of hemagglutinin (H) molecules from<SUP> </SUP>serologically non-cross-reactive H1N1 and H3N2 influenza A viruses,<SUP> </SUP>the aim being to analyze recall CD4<SUP>+</SUP> T cell responses in a virus-induced<SUP> </SUP>respiratory disease. Prime/challenge experiments with these<SUP> </SUP>H1ova and H3ova viruses in H2<SUP>b</SUP> mice gave the predicted, ovalbumin-specific<SUP> </SUP>CD4<SUP>+</SUP> T cell response but showed an unexpectedly enhanced, early<SUP> </SUP>expansion of viral epitope-specific CD8<SUP>+</SUP> T cells in spleen and<SUP> </SUP>a greatly diminished inflammatory process in the virus-infected<SUP> </SUP>respiratory tract. At the same time, the primary antibody response<SUP> </SUP>to the H3N2 challenge virus was significantly reduced, an effect<SUP> </SUP>that has been associated with preexisting neutralizing antibody<SUP> </SUP>in other experimental systems. Analysis of serum from the H1ova-primed<SUP> </SUP>mice showed low-level binding to H3ova but not to the wild-type<SUP> </SUP>H3N2 virus. Experiments with CD4<SUP>+</SUP> T cell-depleted and Ig<SUP>?/?</SUP><SUP> </SUP>mice indicated that this cross-reactive Ig is indeed responsible<SUP> </SUP>for the modified pathogenesis after respiratory challenge. Furthermore,<SUP> </SUP>the effect does not seem to be virus-dose related, although<SUP> </SUP>it does require infection. These findings suggest intriguing<SUP> </SUP>possibilities for vaccination and, at the same time, emphasize<SUP> </SUP>that engineered modifications in viruses may have unintended<SUP> </SUP>immunological consequences.<SUP> </SUP>

    cross-reactivity | vaccines
    <HR align=center width="50%" noShade SIZE=1>Many of the vaccination strategies for promoting cell-mediated<SUP> </SUP>immunity against lethal pathogens such as HIV use other RNA<SUP> </SUP>or DNA viruses that have been engineered to incorporate sequences<SUP> </SUP>encoding foreign peptides (1). Such protocols can involve DNA<SUP> </SUP>priming followed by boosting with the modified vector, which<SUP> </SUP>is generally a high-titer, infectious (but nonreplicating) or<SUP> </SUP>severely attenuated virus. Sometimes two different vectors,<SUP> </SUP>such as vaccinia and adenovirus, are used for the primary and<SUP> </SUP>secondary challenges. Prime/boost studies with selectively mutated<SUP> </SUP>viruses have also facilitated the experimental dissection of,<SUP> </SUP>in particular, virus-specific CD8<SUP>+</SUP> T cell responses. The laboratory<SUP> </SUP>strain A/PR/8/34 (PR8, H1N1) and A/HK/x31 (Hkx31, H3N2) influenza<SUP> </SUP>A viruses have been used extensively for this purpose (2, 3).<SUP> </SUP>
    The preferred site for peptide insertion in the influenza A<SUP> </SUP>viruses has generally been the stalk of the viral neuraminidase<SUP> </SUP>(N) molecule, which can tolerate an additional 40 or so additional<SUP> </SUP>amino acids without obvious functional compromise (4). However,<SUP> </SUP>some molecules do not express in the N, so an alternative protocol<SUP> </SUP>(5) is to modify the globular head of the viral hemagglutinin<SUP> </SUP>(HA or H). This protocol has been used successfully to insert<SUP> </SUP>both CD8<SUP>+</SUP> T cell and B cell epitopes. N and HA are the two principal<SUP> </SUP>glycoproteins expressed on the surface of the influenza A viruses<SUP> </SUP>and, as such, are subject to antibody-mediated selection pressure.<SUP> </SUP>The HA binds to sialic acid and plays a key part in virus entry,<SUP> </SUP>whereas the N has the opposite role of facilitating the release<SUP> </SUP>of new virus progeny.<SUP> </SUP>
    "Antigenic drift" in, particularly, the HA is responsible for<SUP> </SUP>the periodic epidemics associated with, for instance, the Hong<SUP> </SUP>Kong (H3N2) influenza A viruses that have been causing severe<SUP> </SUP>human disease for more than 30 years (5). Many of these natural<SUP> </SUP>variants emerge as a consequence of mutational changes that<SUP> </SUP>modify the globular head of the HA molecule and abrogate or<SUP> </SUP>diminish the extent of neutralization by antibodies generated<SUP> </SUP>as a result of exposure to an earlier iteration of the H3 molecule<SUP> </SUP>(6). More distant influenza strains, such as the H1N1 "human"<SUP> </SUP>viruses or the H5N1 "avian" strains, induce responses that show<SUP> </SUP>no evidence of cross-neutralization, either with each other<SUP> </SUP>or with H3N2 isolates. A mutation in the HA of what may have<SUP> </SUP>originally been a bird pathogen is thought to have contributed<SUP> </SUP>to the extreme pathogenicity of the H1N1 influenza A virus that<SUP> </SUP>killed more than 40 million people during the course of the<SUP> </SUP>1918?1919 pandemic (7).<SUP> </SUP>
    Despite this understanding that glycoprotein structure may be<SUP> </SUP>a major determinant of both antigenicity and pathogenicity,<SUP> </SUP>little thought is generally given to the possibility that changes<SUP> </SUP>other than those that modify fitness (measured by the capacity<SUP> </SUP>to replicate) will have any substantial effect on the endogenous<SUP> </SUP>response to a viral vector. This was certainly the case when<SUP> </SUP>we inserted the coding sequences for an ovalbumin peptide (OVA<SUB>323?339</SUB>)<SUP> </SUP>into the HA molecules of H1N1 and H3N2 influenza A viruses.<SUP> </SUP>The OVA<SUB>323?339</SUB> peptide binds to the H2-IA<SUP>b</SUP> MHC class II<SUP> </SUP>glycoprotein to form the OT-II epitope; therefore, we anticipated<SUP> </SUP>that prime/boost experiments with these two viruses (H1ova and<SUP> </SUP>H3ova) in C57BL/6J H2<SUP>b</SUP> (B6) mice would promote significant clonal<SUP> </SUP>expansion of OT-II-specific CD4<SUP>+</SUP> T cells. The expected result<SUP> </SUP>was achieved, but the surprise was the generation of a cross-reactive,<SUP> </SUP>although weak, antibody response (between H1ova and H3ova) that<SUP> </SUP>modified the characteristics of secondary influenza-specific<SUP> </SUP>CD8<SUP>+</SUP> T cell-mediated immunity. This unpredicted finding has<SUP> </SUP>obvious implications for vaccines based upon viral vectors that<SUP> </SUP>may be subject to preexisting antibody responses within a population.<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> Results </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
    Abstract
    Results
    Discussion
    Materials and Methods
    Acknowledgements
    References
    </TH></TR></TBODY></TABLE>
    Virus Clearance and CD4<SUP>+</SUP> T Cell and Antibody Responses. These H1ova (Fig. 1) and H3ova viruses were generated to analyze<SUP> </SUP>a possible role for OT-II-specific CD4<SUP>+</SUP> T cells (8) in the H3N2<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1N1<SUP> </SUP>influenza A virus prime/boost model that we routinely use to<SUP> </SUP>dissect virus-specific CD8<SUP>+</SUP> T cell-mediated immunity (9). Although<SUP> </SUP>infection of na?ve B6 mice with either the H1ova or H3ova<SUP> </SUP>viruses did not induce a detectable, acute OT-II-specific CD4<SUP>+</SUP><SUP> </SUP>T cell response (data not shown), it was apparent that the memory<SUP> </SUP>compartment had been primed because significant numbers of OT-II-specific<SUP> </SUP>T cells were found in spleen after a secondary challenge (Fig. 2A).<SUP> </SUP>Furthermore, although the engineered H3ova virus grows<SUP> </SUP>to much the same extent as the wild-type (wt) H3 virus in the<SUP> </SUP>lungs of na?ve mice (H3ova, 6.6 ? 0.54; H3wt, 7.3<SUP> </SUP>? 0.14), those that had been injected i.p. with the H1ova<SUP> </SUP>virus 6 weeks earlier cleared a subsequent H3ova infection more<SUP> </SUP>rapidly than the H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt controls (Fig. 2B).<SUP> </SUP>
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    </NOBR> </TD><TD vAlign=top align=left>Fig. 1. Model depicting the insertion of the OVA<SUB>323?339</SUB> peptide (red) into the globular head of H1 (yellow). The engineered H3 is essentially identical. The insertion site is in the loop near antigenic site B. Although the structure of the loop with the OVA peptide is hypothetical, it illustrates why the change is likely to promote antibody binding. This model was generated by S. White (St. Jude Children?s Research Hospital).
    </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER><SUP></SUP>
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    </NOBR> </TD><TD vAlign=top align=left>Fig. 2. The response to secondary challenge, measured for five mice per group. (A) Quantification of OT-II-specific CD4<SUP>+</SUP> T cells by ELISPOT analysis of spleen on day 8. (B) Virus titers in lung as determined by Madin?Darby canine kidney (MDCK) cell plaque assay. (C) Serum antibody titers measured by antigen-specific ELISA on days 2, 5, and 8 of secondary challenge. (D) Absence of a CD8<SUP>+</SUP> T cell response on day 8 after homologous challenge of H3wt-infected mice. Results are expressed as mean ? SE (n = 5); <SUB>*</SUB>, P < 0.05.
    </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>Given that cross-reactive helper T cells had been primed (Fig. 2B),<SUP> </SUP>an obvious question was whether priming in any way enhanced<SUP> </SUP>the antibody response to the engineered H3ova virus. Paradoxically,<SUP> </SUP>both the virus-specific (Fig. 2C) and the H1 (data not shown)<SUP> </SUP>antibody responses were substantially reduced for the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova<SUP> </SUP>combination. This was exactly opposite of the predicted result<SUP> </SUP>but was reminiscent of experiments with other model systems<SUP> </SUP>in which a small amount of preexisting virus-specific Ig can<SUP> </SUP>be shown to diminish the development of a na?ve B cell<SUP> </SUP>response (10). The next step, therefore, was to look at serum<SUP> </SUP>from mice that had been primed only with the H1ova virus. The<SUP> </SUP>results indicated (Table 1) that exposure to the modified H1<SUP> </SUP>of the H1ova virus (Fig. 1) generated a low-level antibody response<SUP> </SUP>specific for the comparably engineered H3 of the H3ova virus.<SUP> </SUP>
    <SUP></SUP>
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    <CENTER><TABLE cellSpacing=0 cellPadding=0 width="95%"><TBODY><TR bgColor=#e1e1e1><TD><TABLE cellSpacing=2 cellPadding=2><TBODY><TR bgColor=#e1e1e1><TD vAlign=top align=middle bgColor=#ffffff>View this table:
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    </NOBR> </TD><TD vAlign=top align=left>Table 1. Serological cross-reactivity for the H3 viruses after priming with the H1 viruses
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    Consequences for the CD8<SUP>+</SUP> T Cell Response. What are the effects of primed CD4<SUP>+</SUP> T help (Fig. 2A) (11) and<SUP> </SUP>low-level cross-reactive Ig on secondary CD8<SUP>+</SUP> T cell responses?<SUP> </SUP>On one hand, high-titer neutralizing antibody is known to block<SUP> </SUP>CD8<SUP>+</SUP> T cell stimulation, presumably because elimination of the<SUP> </SUP>input inoculum (12) prevents epitope expression on antigen-presenting<SUP> </SUP>cells. This suppressive effect can be seen for the homologous<SUP> </SUP>H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H3wt challenge shown here (Fig. 2D). On the other hand,<SUP> </SUP>partially cross-reactive influenza HA-specific antibody does<SUP> </SUP>allow some reduced clonal expansion after secondary challenge<SUP> </SUP>(12). The low-titer HA-OT-II-specific Ig (Table 1) generated<SUP> </SUP>in the current experimental system does not significantly inhibit<SUP> </SUP>the initial phase of virus growth in the lung (day 2, Fig. 2B),<SUP> </SUP>so a secondary virus-specific CD8<SUP>+</SUP> T cell response would be<SUP> </SUP>expected.<SUP> </SUP>
    In fact, the development of the various virus-specific CD8<SUP>+</SUP><SUP> </SUP>T cell sets in spleen after the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova challenge seemed<SUP> </SUP>to be significantly enhanced for all except the PB1-F2 epitope<SUP> </SUP>(Fig. 3 A?C). Even so, although this increase was apparent<SUP> </SUP>at days 2 and 5 (Fig. 3A and B), the NP, PA, and PB1 responses<SUP> </SUP>for the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova and H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt challenges measured on day 8<SUP> </SUP>were equivalent in magnitude (Fig. 3C). Paradoxically, despite<SUP> </SUP>the apparently enhanced kinetics in lymphoid tissue, the recruitment<SUP> </SUP>of virus-specific CD8<SUP>+</SUP> T cells to the infected lung was greatly<SUP> </SUP>diminished for the H1ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H3ova challenge (Fig. 3D).<SUP> </SUP>
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    </NOBR> </TD><TD vAlign=top align=left>Fig. 3. Measuring epitope-specific CD8<SUP>+</SUP> T cell responses in spleen on day 2 (A), day 5 (B), and day 8 (C) and bronchoalveolar lavage (BAL) on day 5 (D) for the H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt and H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova challenges. Influenza virus epitopes are as follows: NP, nucleoprotein D<SUP>b</SUP>NP<SUB>366-374</SUB>; PA and PB1, RNA polymerase subunits PA (D<SUP>b</SUP>PA<SUB>224-233</SUB>) and PB1 (K<SUP>b</SUP>PB1<SUB>703-711</SUB>); F2, an alternatively spliced PB1 (D<SUP>b</SUP>PB1-F2<SUB>62-70</SUB>). Results were determined by intracellular cytokine (IFN-) staining and are expressed as mean ? SE (n = 5); <SUB>*</SUB>, P < 0.05. Experiments on each day were repeated at least twice.
    </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>Possible Virus Dose Effects. The immediate interpretation of the decreased localization of<SUP> </SUP>virus-specific CD8<SUP>+</SUP> T cells to the infected lung (Fig. 3D) was<SUP> </SUP>that it might reflect substantial neutralization (Table 1) of<SUP> </SUP>the inoculum at the time of respiratory challenge, although<SUP> </SUP>such an effect was not apparent from the lung virus titers measured<SUP> </SUP>on day 2 (Fig. 2B). However, although both the generation of<SUP> </SUP>epitope-specific CD8<SUP>+</SUP> T cells in the lymphoid tissue (spleen,<SUP> </SUP>Fig. 4A and B) and subsequent localization to the bronchoalveolar<SUP> </SUP>lavage (BAL) (Fig. 4 C and D) were greatly diminished in the<SUP> </SUP>mice given a low-titer inoculum (Fig. 4, compare A with B and<SUP> </SUP>C with D), dropping the intranasal (i.n.) dose 100-fold (Fig. 4<SUP> </SUP>A?D) reproduced the differential spleen/BAL relationships<SUP> </SUP>for the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova and H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt challenges seen previously on<SUP> </SUP>day 8 (Fig. 3A?D). The minimal inflammatory response in<SUP> </SUP>the lungs of the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova mice is not, therefore, due to a<SUP> </SUP>reduced virus challenge.<SUP> </SUP>
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    </NOBR> </TD><TD vAlign=top align=left>Fig. 4. Epitope-specific CD8<SUP>+</SUP> T cell responses in spleen (A and B) and BAL (C and D) after high-dose [10<SUP>6</SUP> egg 50% infectious dose (EID<SUB>50</SUB>)] (A and C) or low-dose (10<SUP>4</SUP> EID<SUB>50</SUB>) (B and D) secondary intranasal (i.n.) challenge. NS2, nonstructural protein 2. Representative data from two independent experiments are shown. Results were determined by intracellular cytokine staining on day 8 after challenge and are expressed as mean ? SE (n = 5); <SUB>*</SUB>, P < 0.05.
    </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>The next question was whether the apparent enhancement of the<SUP> </SUP>response in the spleen on day 5 (Fig. 3B) depended on virus<SUP> </SUP>replication. Productive infection with the influenza A viruses<SUP> </SUP>requires the presence of an enzyme that cleaves the viral HA<SUP> </SUP>but is restricted in distribution to the mouse respiratory tract<SUP> </SUP>(13). After i.p. exposure, infected cells will produce new protein<SUP> </SUP>but no progeny virus. The i.n. challenge experiment was thus<SUP> </SUP>repeated with high-dose [10<SUP>6</SUP> egg 50% infective dose (EID<SUB>50</SUB>)],<SUP> </SUP>heat-inactivated virus, which is known to prime a CD8<SUP>+</SUP> T cell<SUP> </SUP>response (14) and for an i.p. boost with live virus. In both<SUP> </SUP>cases (data not shown), the enhanced response observed in the<SUP> </SUP>spleen on day 5 after i.n. challenge with infectious virus (Fig. 3B)<SUP> </SUP>was not reproduced.<SUP> </SUP>
    Effect of CD4<SUP>+</SUP> T Cell and Antibody Responses. The increase in spleen CD8<SUP>+</SUP> T cell numbers seen on day 5 (Fig. 3B)<SUP> </SUP>for the secondary H3ova challenge is absolutely dependent<SUP> </SUP>on prior exposure (H1ova priming, Fig. 5A) to the OVA-modified<SUP> </SUP>H1 molecule. Priming with H1wt and challenging with H3ova gives<SUP> </SUP>an equivalent response to challenging with H3wt. This response<SUP> </SUP>includes the full expansion of the BAL response (data not shown),<SUP> </SUP>providing further evidence that the introduced epitope does<SUP> </SUP>not significantly affect the fitness of the virus and proving<SUP> </SUP>the requirement for memory in the observed modulation of the<SUP> </SUP>response. Does this response reflect the concurrent restimulation<SUP> </SUP>of primed, OT-II-specific CD4<SUP>+</SUP> helper T cells, or is it in some<SUP> </SUP>way related to the fact that low-level, cross-reactive antibody<SUP> </SUP>(Table 1) is present from the time of secondary challenge? Eliminating<SUP> </SUP>the CD4<SUP>+</SUP> T cells by treatment with the GK1.5 monoclonal antibody<SUP> </SUP>to CD4 decreased the overall magnitude of the response but did<SUP> </SUP>not obviously change the differential epitope-specific CD8<SUP>+</SUP><SUP> </SUP>T cell response profiles (Fig. 3B and Fig. 5A) found for the<SUP> </SUP>H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt and H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova challenges (Fig. 5 B and C). Thus, although<SUP> </SUP>CD4<SUP>+</SUP> T cells may well be needed for the development of the cross-reactive<SUP> </SUP>antibody found in the H1ova-immune mice, they are not required<SUP> </SUP>for the effect on the CD8<SUP>+</SUP> T cell response (Fig. 3B and Fig. 5A)<SUP> </SUP>seen after the H3ova challenge. Furthermore, the pattern<SUP> </SUP>of early, differential virus clearance on day 5 was not modified<SUP> </SUP>for CD4-depleted or CD8-depleted H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova-primed mice (data<SUP> </SUP>not shown). There is a substantial body of earlier evidence<SUP> </SUP>that the influenza A viruses can be controlled by either CD8<SUP>+</SUP><SUP> </SUP>T cell or CD4<SUP>+</SUP> T help/antibody response (15, 16).<SUP> </SUP>
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    </NOBR> </TD><TD vAlign=top align=left>Fig. 5. Splenic enhancement phenotype is dependent on a primed B-cell response. (A) The enhanced spleen response is seen only for the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova combination. H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt and H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova infections produce responses equivalent to H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt. (B and C) Depleting the CD4<SUP>+</SUP> T cells by treatment with the GK1.5 monoclonal antibody (C) every second day from day 3 before secondary challenge (30) causes some decrease in the overall magnitude of the response vs. undepleted mice (B) but does not change the enhancement characteristic of the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova challenge. These data are representative of three independent experiments. The percentage of CD4<SUP>+</SUP> T cells found by flow cytometric analysis of spleen was always <2% in the depleted mice. (D) The response in Ig<SUP>?/?</SUP> ?MT (B cell knockout) mice after secondary challenge with the indicated viruses. All results were measured by intracellular cytokine staining on day 5 and are expressed as mean ? SE (n = 5); <SUB>*</SUB>, P < 0.05.
    </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></CENTER>It seems possible, then, that this effect (Fig. 3B and Fig. 5<SUP> </SUP>C and D) is mediated by low-level, cross-reactive antibody<SUP> </SUP>that does not require CD4<SUP>+</SUP> T cell help after secondary exposure<SUP> </SUP>(Fig. 5 B and C). The fact that repeating the basic analysis<SUP> </SUP>in Ig<SUP>?/?</SUP> ?MT mutants did not reproduce the<SUP> </SUP>differentials found for the H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt and H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova prime/boost<SUP> </SUP>in Ig<SUP>+/+</SUP> B6 mice (Fig. 5D) supports this interpretation. The<SUP> </SUP>overall magnitude of the response in the Ig<SUP>?/?</SUP> ?MTs<SUP> </SUP>was greatly reduced because of their much smaller spleen size,<SUP> </SUP>but the day 5 responses for the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova and H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt combinations<SUP> </SUP>were not significantly different (Fig. 5D). Moreover, equivalent<SUP> </SUP>BAL responses were measured in these mice after the H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt<SUP> </SUP>and H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova challenges (data not shown).<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> Discussion </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
    Abstract
    Results
    Discussion
    Materials and Methods
    Acknowledgements
    References
    </TH></TR></TBODY></TABLE>
    By using a prime/challenge system in which two serodistinct<SUP> </SUP>viruses have been engineered to contain a short identical sequence,<SUP> </SUP>we have shown that a cross-reactive antibody response that does<SUP> </SUP>not fully neutralize infection allows the development of a full<SUP> </SUP>CD8<SUP>+</SUP> T cell response and leads to the accumulation of antigen-specific<SUP> </SUP>T cells in the spleen rather than at the site of infection.<SUP> </SUP>These findings bear upon the important question of how preexisting<SUP> </SUP>antibody may affect the efficacy of viral vector-based vaccines.<SUP> </SUP>In this case, in the presence of nonsterilizing partial immunity,<SUP> </SUP>a robust CD8<SUP>+</SUP> T cell response was generated although not at<SUP> </SUP>the site of infection. This is an important consideration for<SUP> </SUP>the screening of the effectiveness of viral vectors that may<SUP> </SUP>be subject to preexisting immunity within a population. Furthermore,<SUP> </SUP>these results suggest a new vaccine design strategy in which<SUP> </SUP>a weak cross-reactive antibody response is generated to boost<SUP> </SUP>the generation of CD8<SUP>+</SUP> T cell responses to a viral vector while<SUP> </SUP>limiting any significant pathology of the vector itself.<SUP> </SUP>
    Prior experiments with conventional HA-specific monoclonal antibodies<SUP> </SUP>suggest that such low-level protection can indeed occur (15).<SUP> </SUP>The HAova-specific antibodies do not bind to native OVA (in<SUP> </SUP>ELISA, data not shown), and it seems that a new (although minimal)<SUP> </SUP>"influenza HA" antibody epitope has been generated. The Ig-mediated<SUP> </SUP>effect does not seem to be operating through direct neutralization<SUP> </SUP>of the input inoculum, perhaps reflecting that a measure of<SUP> </SUP>virus-induced damage (or endothelial activation) must occur<SUP> </SUP>in the respiratory tract before the serum HAova-specific Ig<SUP> </SUP>is able to access the luminal, type 1 respiratory epithelium<SUP> </SUP>that supports influenza virus growth (17).<SUP> </SUP>
    Despite the possibility that a measure of virus-induced damage<SUP> </SUP>to the lung is important in the pathogenesis of this infectious<SUP> </SUP>process, it is also the case that the extent of inflammatory<SUP> </SUP>pathology is minimal in the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova-primed mice. The very<SUP> </SUP>small numbers of epitope-specific CD8<SUP>+</SUP> T cells recovered from<SUP> </SUP>this site are simply a reflection of the fact that the magnitude<SUP> </SUP>of mononuclear cell invasion into the infected lung is massively<SUP> </SUP>reduced. Although we present the data as epitope-specific CD8<SUP>+</SUP><SUP> </SUP>T cell counts, the immunodominant D<SUP>b</SUP>NP<SUB>366?374</SUB>-specific<SUP> </SUP>set (18) was always the most prevalent influenza-specific component<SUP> </SUP>in the BAL population recovered from the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova mice, indicating<SUP> </SUP>that, as is generally the case, there is no selectivity in recruitment<SUP> </SUP>to the site of pathology.<SUP> </SUP>
    The minimal inflammatory process may also explain the diminished<SUP> </SUP>response to the PB1-F2 epitope. The lack of clonal expansion<SUP> </SUP>for the PB1-F2-specific set suggests that these T cells are<SUP> </SUP>not primed in the same "window" as the NP, PA, and PB1 responses,<SUP> </SUP>perhaps reflecting that PB1-F2 is not a component of the virus<SUP> </SUP>particle but instead is generated from an alternative reading<SUP> </SUP>frame that is primarily expressed subsequent to productive infection<SUP> </SUP>(19). It is also likely that, because there will be both less<SUP> </SUP>damage and decreased cytokine production in the infected lung,<SUP> </SUP>the transfer of activated dendritic cells from the respiratory<SUP> </SUP>tract to the regional lymphoid tissue via afferent lymph may<SUP> </SUP>be substantially diminished for the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova versus the H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt<SUP> </SUP>challenge (20). However, it is not known whether differential<SUP> </SUP>dendritic cell activation and/or availability has any particular<SUP> </SUP>effect for the PB1-F2 epitope.<SUP> </SUP>
    The fact that the apparent enhanced response kinetics for influenza-specific<SUP> </SUP>CD8<SUP>+</SUP> T cell epitopes (other than PB1-F2) in spleen was seen<SUP> </SUP>only after i.n. challenge with infectious virus may simply reflect<SUP> </SUP>the delayed exit of immune T cells from the spleen. The implication<SUP> </SUP>is therefore that, in a situation in which there is a "normal"<SUP> </SUP>level of inflammatory process in the target organ, restimulated<SUP> </SUP>memory T cells are moving out of the lymphoid tissue as early<SUP> </SUP>as day 2 after infection. This hypothesis implies that between<SUP> </SUP>these two compartments there is "cross talk" which must, presumably,<SUP> </SUP>be mediated through circulating cytokines generated in the site<SUP> </SUP>of pathology.<SUP> </SUP>
    It is often suggested that antibody and CD8<SUP>+</SUP> T cell responses<SUP> </SUP>are in direct opposition because both are antigen-dependent<SUP> </SUP>and because reduced availability (due to neutralization) would<SUP> </SUP>lead to diminished CD8<SUP>+</SUP> T cell numbers (21). This suggestion<SUP> </SUP>has caused some concerns about the usefulness of viral vectors<SUP> </SUP>where preexisting antibody might be circulating in the population.<SUP> </SUP>Instead, we found that the overall size of the CD8<SUP>+</SUP> T cell response<SUP> </SUP>is not altered by (at least) low level, cross-reactive antibody.<SUP> </SUP>In other model systems, particularly in lymphocytic choriomeningitis<SUP> </SUP>and respiratory syncytial viruses, CD8<SUP>+</SUP> T cell responses expand<SUP> </SUP>even in the presence of strongly neutralizing Ig, although high<SUP> </SUP>virus titers are necessary and T cell proliferation is thought<SUP> </SUP>to occur at the site of pathology, producing the pathological<SUP> </SUP>changes characteristic of primary challenge (22, 23). The data<SUP> </SUP>from the H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova challenge indicate that the clonal expansion<SUP> </SUP>of CD8<SUP>+</SUP> T cells in lymphoid tissue is not (except for PB1-F2)<SUP> </SUP>directly tuned to the degree of inflammation in an anatomically<SUP> </SUP>remote target site such as the lung and, even in the secondary<SUP> </SUP>response, follows a predetermined program.<SUP> </SUP>
    There is also a general lesson to be learned from this analysis.<SUP> </SUP>As with genetically manipulated mice (24), it is unwise to come<SUP> </SUP>to any substantial conclusions concerning the possible effects<SUP> </SUP>of mutating, or otherwise modifying, an infectious virus for<SUP> </SUP>immunity without making a detailed, multiparameter analysis.<SUP> </SUP>If the current experiment had looked only at the day 5 response<SUP> </SUP>in spleen and virus clearance from the lung of infected B6 mice,<SUP> </SUP>the conclusion might well have been reached that priming the<SUP> </SUP>OT-II-specific CD4<SUP>+</SUP> set leads directly to a more substantial<SUP> </SUP>virus-specific CD8<SUP>+</SUP> T cell response that promotes rapid recovery.<SUP> </SUP>This is one of the many reasons, including safety (25), that,<SUP> </SUP>where possible, any proposed use of replicating, engineered<SUP> </SUP>"vaccine" reagents in humans should be preceded by a detailed<SUP> </SUP>analysis of mechanism in appropriate laboratory animal models.<SUP> </SUP>
    The other speculation arising from these studies is that it<SUP> </SUP>may be possible to modify the key antigenic sites of influenza<SUP> </SUP>A viruses to promote low-level antibody responses that, although<SUP> </SUP>they may not prevent infection, could provide a measure of protection,<SUP> </SUP>enhance recovery, and allow the full expansion of the CD8<SUP>+</SUP> T<SUP> </SUP>cell response. Preliminary experiments (data not shown) in which<SUP> </SUP>we mutated the globular head of the H1 glycoprotein to mimic<SUP> </SUP>a peptide from the H3 molecule that is known to stimulate CD4<SUP>+</SUP><SUP> </SUP>T cells seemed to give an effect similar to that described here<SUP> </SUP>for the OVA-modified viruses. Can viral glycoproteins be engineered<SUP> </SUP>to give cross-reactive antibody responses that might not normally<SUP> </SUP>be stimulated after natural infection but may nonetheless provide<SUP> </SUP>some protection against, say, a previously unencountered, virulent<SUP> </SUP>influenza A virus?<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> Materials and Methods </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
    Abstract
    Results
    Discussion
    Materials and Methods
    Acknowledgements
    References
    </TH></TR></TBODY></TABLE>
    Viruses, Mice, and Sampling. Reverse genetics protocols (26, 27) were used to insert the<SUP> </SUP>OVA<SUB>323?339</SUB> sequence (ISQAVHAAHAEINEAGR) after the glycines<SUP> </SUP>at residue 173 of the H1 (PR8, H1N1) and 174 of the H3 (the<SUP> </SUP>A/Aichi HA of HKx31) HA glycoproteins (Fig. 1). No residues<SUP> </SUP>were removed. The resultant H1ova and H3ova viruses were rescued<SUP> </SUP>in 10-day-old embryonated chicken eggs after the engineered<SUP> </SUP>plasmids were transfected into cocultures of 293 T cells and<SUP> </SUP>MDCK cells. Female B6 mice were purchased from The Jackson Laboratory<SUP> </SUP>and Ig<SUP>?/?</SUP> ?MT mice were bred at St. Jude Children?s<SUP> </SUP>Research Hospital. All mice were held under specific pathogen-free<SUP> </SUP>conditions and generally primed by i.p. injection with 10<SUP>8</SUP> EID<SUB>50</SUB><SUP> </SUP>of wt H1 or H1ova viruses. They were then left for 6?8<SUP> </SUP>weeks and, after anesthesia by i.p. injection of 2,2,2-tribromoethanol<SUP> </SUP>(Avertin), challenged i.n. with 10<SUP>6</SUP> EID<SUB>50</SUB> of the H3wt or H3ova<SUP> </SUP>viruses. The mice were anesthetized again at the time of sampling<SUP> </SUP>and exsanguinated by sectioning the axillary artery. Inflammatory<SUP> </SUP>cell populations were recovered from the infected respiratory<SUP> </SUP>tract by BAL, followed by removal of the spleen to prepare single-cell<SUP> </SUP>suspensions for lymphocyte analysis and to prepare the lungs<SUP> </SUP>for later virus plaque assay.<SUP> </SUP>
    Measuring Virus-Specific CD8<SUP>+</SUP> T Cells. Epitope-specific CD8<SUP>+</SUP> T cell responses were analyzed by the<SUP> </SUP>intracellular cytokine staining flow cytometry assay. Inflammatory<SUP> </SUP>cell populations recovered from the pneumonic lung by BAL were<SUP> </SUP>depleted of macrophages by incubation on plastic for 1 h at<SUP> </SUP>37?C. Spleen and BAL lymphocytes were then incubated with<SUP> </SUP>1 ?M peptide for 5 h in the presence of 10 ?g/ml<SUP> </SUP>brefeldin A, fixed with formaldehyde, and stained with CD8 allophycocyanin<SUP> </SUP>(53-6.7), IFN- phycoerythrin (Pharmingen, catalog no. 554412),<SUP> </SUP>and TNF- FITC (catalog no. 554418). Autofluorescence was gated<SUP> </SUP>out by using the FL3 channel. Data were acquired on a Becton<SUP> </SUP>Dickinson FACSCalibur and analyzed by using CELLQUEST software.<SUP> </SUP>The epitope-specific responses measured were to D<SUP>b</SUP>NP<SUB>366?374</SUB>,<SUP> </SUP>D<SUP>b</SUP>PA<SUB>224?233</SUB>, D<SUP>b</SUP>PB1-F2<SUB>62?70</SUB>, K<SUP>b</SUP>PB1<SUB>703?711</SUB>,<SUP> </SUP>and K<SUP>b</SUP>NS2<SUB>114?121</SUB> (2, 28).<SUP> </SUP>
    Virus Titration. Lung titers were determined by using plaque assay on MDCK cells.<SUP> </SUP>Near-confluent 25-cm<SUP>2</SUP> monolayers were infected with serial dilutions<SUP> </SUP>of lung homogenate (1 ml) for 1 h at 37?C then washed with<SUP> </SUP>PBS and 3 ml of MEM containing 1 mg/ml L-1-tosylamido-2-phenylethyl<SUP> </SUP>chloromethyl ketone-treated trypsin (Worthington). Agarose (0.8%)<SUP> </SUP>was added and the cultures were incubated at 37?C under<SUP> </SUP>a 5% CO<SUB>2</SUB> atmosphere for 72 h. Plaques were visualized with crystal<SUP> </SUP>violet.<SUP> </SUP>
    Counting CD4<SUP>+</SUP> T Cells. An established ELISPOT assay (12) was used to quantify OT-II-specific<SUP> </SUP>IFN--producing CD4<SUP>+</SUP> T cells in spleen after stimulation with<SUP> </SUP>virus-infected, antigen-presenting cells. Cells were exposed<SUP> </SUP>to the OVA<SUB>323?339</SUB> peptide or no peptide, and the number<SUP> </SUP>of IFN- producers was measured as spots per 10<SUP>6</SUP> input cells<SUP> </SUP>after 48 h at 37?C.<SUP> </SUP>
    Antibody Assays. Standard hemagglutination-inhibition, neutralization, and ELISA<SUP> </SUP>protocols were followed (29). The ELISA used virus dried onto<SUP> </SUP>plates as an antigen. Mouse sera were treated overnight with<SUP> </SUP>Vibrio cholerae receptor-destroying enzyme (Denka-Seiken, Tokyo),<SUP> </SUP>heat inactivated for 30 min at 56?C, then assayed by a standard<SUP> </SUP>hemagglutination-inhibition protocol (29) by using 4 HA units<SUP> </SUP>of virus. Sera were also heat inactivated (as above) for the<SUP> </SUP>plaque neutralization assays and only heated (no enzyme treatment)<SUP> </SUP>for the egg neutralization assays, diluted serially in PBS,<SUP> </SUP>mixed with 100 EID<SUB>50</SUB> of virus, incubated for 30 min at 4?C,<SUP> </SUP>then overlaid on MDCK monolayers or injected into 10-day-old<SUP> </SUP>embryonated hen?s eggs. After 48 h at 37?C, the eggs<SUP> </SUP>were chilled overnight and harvested the next day. Virus that<SUP> </SUP>had not been neutralized was detected by hemagglutination (eggs)<SUP> </SUP>or as cytopathic effects (MDCK).<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> Acknowledgements </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
    Abstract
    Results
    Discussion
    Materials and Methods
    Acknowledgements
    References
    </TH></TR></TBODY></TABLE>
    We thank Dr. Stephen White for generating the model in Fig. 1.<SUP> </SUP>This work was supported by U.S. Public Health Service Grants<SUP> </SUP>AI29579 and CA21765 (to P.C.D.) and AI065097 (to P.G.T.) and<SUP> </SUP>by American Lebanese Syrian Associated Charities at St. Jude<SUP> </SUP>Children?s Research Hospital. P.C.D. is a Burnet Fellow<SUP> </SUP>of the Australian National Health and Medical Research Council.<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> Footnotes </TH></TR></TBODY></TABLE>

    Abbreviations: OVA, ovalbumin; HA or H, hemagglutinin; N, viral neuraminidase; BAL, bronchoalveolar lavage; MDCK, Madin?Darby canine kidney; EID<SUB>50</SUB>, egg 50%infective dose; i.n., intranasal(ly); B6, C57BL/6J H2<SUP>b</SUP> mice; wt, wild type.
    <!-- null --><SUP></SUP>To whom correspondence should be addressed. E-mail: peter.doherty@stjude.org<SCRIPT type=text/javascript><!-- var u = "peter.doherty", d = "stjude.org"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></SCRIPT>
    <!-- null -->Contributed by Peter C. Doherty, December 26, 2005<SUP> </SUP>
    <!-- null -->Author contributions: P.G.T., S.A.B., R.J.W., and P.C.D. designed<SUP> </SUP>research; P.G.T., S.A.B., W.Y., and J.S. performed research;<SUP> </SUP>P.G.T. and R.J.W. contributed new reagents/analytic tools; P.G.T.,<SUP> </SUP>W.Y., and J.S. analyzed data; and P.G.T. and P.C.D. wrote the<SUP> </SUP>paper.<SUP> </SUP>
    <!-- null -->Conflict of interest statement: No conflicts declared.<SUP> </SUP>
    ? 2006 by The National Academy of Sciences of the USA
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    <TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> References </TH></TR></TBODY></TABLE><TABLE cellPadding=5 align=right border=1><TBODY><TR><TH align=left>Top
    Abstract
    Results
    Discussion
    Materials and Methods
    Acknowledgements
    References
    </TH></TR></TBODY></TABLE>
    1. <!-- null --><LI value=1>Stephenson, J. R. (2001) Curr. Pharm. Biotechnol 2, 47?76.<!-- HIGHWIRE ID="103:8:2764:1" -->[CrossRef][Medline] <!-- /HIGHWIRE --><!-- null --><LI value=2>Belz, G. T., Xie, W. & Doherty, P. C. (2001) J. Immunol 166, 4627?4633.<!-- HIGHWIRE ID="103:8:2764:2" --><NOBR>[Abstract/Free Full Text]</NOBR><!-- /HIGHWIRE --><!-- null --> <LI value=3>Riberdy, J. M., Christensen, J. P., Branum, K. & Doherty, P. C. (2000) J. Virol 74, 9762?9765.<!-- HIGHWIRE ID="103:8:2764:3" --><NOBR>[Abstract/Free Full Text]</NOBR><!-- /HIGHWIRE --><!-- null --> <LI value=4>Luo, G., Chung, J. & Palese, P. (1993) Virus Res 29, 141?153.<!-- HIGHWIRE ID="103:8:2764:4" -->[CrossRef][ISI][Medline] <!-- /HIGHWIRE --><!-- null --><LI value=5>Both, G. W., Sleigh, M. J., Cox, N. J. & Kendal, A. P. (1983) J. Virol 48, 52?60.<!-- HIGHWIRE ID="103:8:2764:5" --><NOBR>[Abstract/Free Full Text]</NOBR><!-- /HIGHWIRE --><!-- null --> <LI value=6>Wiley, D. C., Wilson, I. A. & Skehel, J. J. 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  • #2
    Figure 1



    Fig. 1. Model depicting the insertion of the OVA<SUB>323?339</SUB> peptide (red) into the globular head of H1 (yellow). The engineered H3 is essentially identical. The insertion site is in the loop near antigenic site B. Although the structure of the loop with the OVA peptide is hypothetical, it illustrates why the change is likely to promote antibody binding. This model was generated by S. White (St. Jude Children?s Research Hospital).

    Comment


    • #3
      Figure 2



      Fig. 2. The response to secondary challenge, measured for five mice per group. (A) Quantification of OT-II-specific CD4<SUP>+</SUP> T cells by ELISPOT analysis of spleen on day 8. (B) Virus titers in lung as determined by Madin?Darby canine kidney (MDCK) cell plaque assay. (C) Serum antibody titers measured by antigen-specific ELISA on days 2, 5, and 8 of secondary challenge. (D) Absence of a CD8<SUP>+</SUP> T cell response on day 8 after homologous challenge of H3wt-infected mice. Results are expressed as mean ? SE (n = 5); <SUB>*</SUB>, P < 0.05.

      Comment


      • #4
        Table 1

        Table 1. Serological cross-reactivity for the H3 viruses after priming with the H1 viruses
        <TABLE border=0><TBODY><TR><TD><TABLE cellSpacing=10 cellPadding=0 width="100%"><TBODY><TR><TD colSpan=5><HR noShade SIZE=1></TD></TR><TR><TD vAlign=top align=left rowSpan=3>Serological test</TD><TD vAlign=top align=middle colSpan=4>No. of positive mice/no. of mice primed
        <HR noShade SIZE=1></TD></TR><TR><TD vAlign=top align=middle colSpan=2>H1wt serum
        <HR noShade SIZE=1></TD><TD vAlign=top align=middle colSpan=2>H1ova serum
        <HR noShade SIZE=1></TD></TR><TR><TD vAlign=top align=middle>Against H3wt virus</TD><TD vAlign=top align=middle>Against H3ova virus</TD><TD vAlign=top align=middle>Against H3wt virus</TD><TD vAlign=top align=middle>Against H3ova virus</TD></TR><TR><TD colSpan=5><HR></TD></TR><TR><TD vAlign=top align=left>Hemagglutination inhibition</TD><TD vAlign=top align=middle>0/5</TD><TD vAlign=top align=middle>0/5</TD><TD vAlign=top align=middle>0/4</TD><TD vAlign=top align=middle>4/4 1:35 average titer</TD></TR><TR><TD vAlign=top align=left>MDCK virus neutralization</TD><TD vAlign=top align=middle>0/6</TD><TD vAlign=top align=middle>0/6</TD><TD vAlign=top align=middle>0/6</TD><TD vAlign=top align=middle>3/6 1:100 average titer of positives</TD></TR><TR><TD vAlign=top align=left>Embryonated hen egg neutralization</TD><TD vAlign=top align=middle>0/5</TD><TD vAlign=top align=middle>0/5</TD><TD vAlign=top align=middle>1/4 >1:40 average titer of positives</TD><TD vAlign=top align=middle>4/4 >1:40 average titer</TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
        <!-- tblfn -->The hemagglutination inhibition and neutralization tests utilized serum taken at least 7 weeks after i.p. priming with the H1wt and H1ova viruses.

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        • #5
          Table 3



          Fig. 3. Measuring epitope-specific CD8<SUP>+</SUP> T cell responses in spleen on day 2 (A), day 5 (B), and day 8 (C) and bronchoalveolar lavage (BAL) on day 5 (D) for the H3wt<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt and H3ova<IMG alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova challenges. Influenza virus epitopes are as follows: NP, nucleoprotein D<SUP>b</SUP>NP<SUB>366-374</SUB>; PA and PB1, RNA polymerase subunits PA (D<SUP>b</SUP>PA<SUB>224-233</SUB>) and PB1 (K<SUP>b</SUP>PB1<SUB>703-711</SUB>); F2, an alternatively spliced PB1 (D<SUP>b</SUP>PB1-F2<SUB>62-70</SUB>). Results were determined by intracellular cytokine (IFN-) staining and are expressed as mean ? SE (n = 5); <SUB>*</SUB>, P < 0.05. Experiments on each day were repeated at least twice.

          Comment


          • #6
            Figure 4



            Fig. 4. Epitope-specific CD8<SUP>+</SUP> T cell responses in spleen (A and B) and BAL (C and D) after high-dose [10<SUP>6</SUP> egg 50% infectious dose (EID<SUB>50</SUB>)] (A and C) or low-dose (10<SUP>4</SUP> EID<SUB>50</SUB>) (B and D) secondary intranasal (i.n.) challenge. NS2, nonstructural protein 2. Representative data from two independent experiments are shown. Results were determined by intracellular cytokine staining on day 8 after challenge and are expressed as mean ? SE (n = 5); <SUB>*</SUB>, P < 0.05.

            Comment


            • #7
              Figure 5




              Fig. 5. Splenic enhancement phenotype is dependent on a primed B-cell response. (A) The enhanced spleen response is seen only for the H3ova<IMG title="->" alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova combination. H3ova<IMG title="->" alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt and H3wt<IMG title="->" alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova infections produce responses equivalent to H3wt<IMG title="->" alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1wt. (B and C) Depleting the CD4<SUP>+</SUP> T cells by treatment with the GK1.5 monoclonal antibody (C) every second day from day 3 before secondary challenge (30) causes some decrease in the overall magnitude of the response vs. undepleted mice (B) but does not change the enhancement characteristic of the H3ova<IMG title="->" alt="->" src="http://www.pnas.org/math/rarr.gif" border=0>H1ova challenge. These data are representative of three independent experiments. The percentage of CD4<SUP>+</SUP> T cells found by flow cytometric analysis of spleen was always <2% in the depleted mice. (D) The response in Ig<SUP>?/?</SUP> ?MT (B cell knockout) mice after secondary challenge with the indicated viruses. All results were measured by intracellular cytokine staining on day 5 and are expressed as mean ? SE (n = 5); <SUB>*</SUB>, P < 0.05.

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