Intranasal administration of peptide vaccine protects human/mouse radiation chimera from influenza infection
Tamar Ben-Yedidia,
Hadar Marcus,
Yair Reisner and
Ruth Arnon
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
Correspondence to:
R. Arnon
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Abstract
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Influenza virus is characterized by frequent and unpredictable changes of the surface glycoproteins which enable the virus to escape the immune system. Approved vaccines which consist of the whole virus or the surface glycoproteins fail to induce broad specificity protection. We have previously reported that a peptide-based experimental recombinant vaccine which includes conserved epitopes of B and T lymphocytes was efficient in mice, leading to cross-strain, long-term protection. In the present study, this approach was adapted for the design of a human vaccine, based on epitopes recognized by the prevalent HLAs. These epitopes were expressed in Salmonella flagellin and tested for their efficacy in human/mouse radiation chimera in which human peripheral blood mononuclear cells (PBMC) are functionally engrafted. The vaccinated mice demonstrated clearance of the virus after challenge and resistance to lethal infection. The production of virus-specific human antibodies was also higher in this group. Control groups of either non-vaccinated, or vaccinated mice which had not been engrafted with the human PBMC, did not exhibit the protective immune response. FACS analysis showed that most human cells in the transplanted mice are CD8+ and CD4+. Hence, it may be concluded: (i) that the protection involves cellular mechanisms, but is most probably accomplished without direct lysis of influenza-infected pulmonary cells by cytotoxic T lymphocytes, but rather via a cytokine-mediated mechanism, (ii) that the human/mouse radiation chimera model may be of some value in the investigation of new vaccines, as an additional tool prior to clinical trials, and (iii) that the synthetic recombinant vaccine can induce a response in the human immune system and confers protection against influenza infection. Further investigation is needed to establish the efficacy of such a peptide vaccine in human subjects.
Keywords: epitope, flagellin, human, influenza, vaccine
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Introduction
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Influenza infection may result in a variety of disease states, ranging from sub-clinical infection through a mild upper respiratory infection and tracheobronchitis to a severe occasionally lethal viral pneumonia. The reasons for this wide spectrum of severity are explained by the site of infection and the immune status of the host. The most important characteristic of influenza, from the immunological point of view, is the rapid, unpredictable changes of the surface glycoproteins, hemagglutinin (HA) and neuraminidase, referred to as antigenic shifts and drifts. These changes lead eventually to the emergence of new influenza strains, that are the cause for almost annual epidemics (13).
Immunization towards influenza virus is limited by this marked antigenic variation of the virus and by the restriction of the infection to the respiratory mucous membranes. The vaccines currently available and licensed are based either on whole inactive virus or on viral surface glycoproteins. These vaccines fail to induce complete, long-term and cross-strain immunity. The use of attenuated live virus could elevate the efficacy of vaccination but such vaccines are not available yet. Another approach that is explored in our laboratory is the use of a peptide-based vaccine. It is known that both B and T lymphocytes react with specific epitopes. Identification of conserved epitopes, shared by multiple viral strains, that stimulate antibody production, Th function and cytotoxic T lymphocytes (CTL) should lead to a more efficient vaccine. Efforts in our laboratory are towards the development of a vaccine preparation including a combination of such epitopes (4).
A synthetic recombinant anti-influenza vaccine based on three epitopes was found in our laboratory to be highly efficient in mice (5). This vaccine included a B cell epitope from the HA 91108 which is conserved in all H3 strains and elicits anti-influenza neutralizing antibodies (6), together with a Th and CTL epitopes from the nucleoprotein (NP 5569 and NP 147158, respectively), which induce MHC-restricted immune responses (4,7). These epitopes were individually expressed in the flagellin of the Salmonella vaccine strain. Intranasal (i.n.) administration of the mixture of the resultant isolated flagella to mice resulted in efficient protection against viral infection (8).
To explore whether this approach can be adapted for the design of a human vaccine, we employed peptide epitopes reactive with human HLAs, and evaluated their effect in a humanized mice model using lethally irradiated BALB/c mice radioprotected with SCID bone marrow and transplanted with human peripheral blood mononuclear cells (PBMC) (9). These mice were shown to accept the engraftment of functioning human PBMC very quickly due to their conditioning regimen. The approach in this model is based on the supralethal conditioning of the recipient mice, to eradicate most of their normal hematopoietic system, followed by radioprotection with a transplant of bone marrow from a SCID donor, which promptly reconstitutes the entire hemopoietic system except for the lymphoid lineage. It is also most significant that the engraftment of human cells in this model does not lead to EpsteinBarr virus lymphoma as reported for SCID mice (10).
In the present study, three T cell epitopes were selected due to their specific recognition by the prevalent HLAs in the Caucasian population (1117). They were included in the vaccine together with the previously described B cell epitope (18). In order to overcome the problem of antigenic variation of the virus, all these epitopes were derived from conserved regions in the virus proteins and, hence, are expected to induce a cross-strain protection. Thus, the vaccine construct includes four epitopes: (i) the CTL epitope comprising residues 335350, which is restricted to A2, A3, Aw68.1 and B37 (16,17,19), (ii) the epitope NP 380393, restricted to B8 and B27 (20,21), (iii) a Th epitope from HA, which resides in the region 307319, which is a `universal' epitope restricted to most class II molecules, including DR1, DR2, DR4, DR5, DR7, DR9 and DR52A (2224), and (iv) the B cell epitope (HA 91108). These epitopes were expressed individually in Salmonella flagellin and the mixture of resultant flagella was used for vaccination of the human/mouse radiation chimera. The new angle in our study is that the vaccine used comprised the combination of four epitopes identified in different studies, and was employed and evaluated in the human/mouse chimera.
We demonstrate that a combination of these epitopes induces a human response specific to the virus when the vaccine was administered to chimeric mice i.n. without any adjuvant. The vaccinated mice were also protected from a lethal infection and their recovery was quicker.
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Methods
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Mice
BALB/c mice (48 weeks old) were obtained from Olac Farms (Bicester, UK) and NOD/SCID mice (46 weeks old) from the Weizmann Institute Animal Breeding Center (Rehovot, Israel). All mice were fed sterile food and acid water containing ciprofloxacin (20 µg/ml).
Conditioning regimen.
BALB/c mice were exposed to a split lethal total body irradiation of 4 Gy followed 3 days later by 10 Gy (10).The source of radiation was a
beam 150-A 60Co (produced by Atomic Energy of Canada, Kanata, Ontario).
Bone marrow cells from NOD/SCID mice (46 weeks old) were obtained according to Levite et al. (25). Recipient irradiated mice were injected with 23x106 SCID bone marrow cells (i.v. in 0.2 ml PBS) 1 day after the second dose of irradiation.
Preparation and transplantation of human peripheral blood lymphocytes
Buffy coats from normal volunteers were layered onto Lymphoprep solution (Nycomed, Oslo, Norway) and spun at 2000 r.p.m. for 20 min. The interlayer was collected, washed twice, counted and resuspended in PBS, pH 7.4, to the desired cell concentration. Human PBMC (70x106 cells in 0.5 ml PBS) were injected i.p. into recipient mice, conditioned as described above 1 day after NOD/SCID bone marrow infusion. Control mice did not receive human PBMC.
Leukapheresis procedure.
Leukapheresis was performed on normal volunteers. Cells were collected by processing 34 l of blood through a Haemonetics V50 over 33.5 h. The leukapheresis product was centrifuged at 1200 r.p.m. for 10 min and the plasma removed.
Influenza virus
The influenza strains A/PR/8/34, A/Japanese/57, A/Texas/1/77 and B/Harbin/7/94 were used. Virus amounts were measured in hemagglutination units (HAU). For immunization, the inactive virus (A/Texas/1/77) purified by sucrose gradient was used. Virus growth and purification were according to standard methods (26). For virus titration, lung samples were homogenized in PBS containing 0.1% BSA and centrifuged in order to remove debris. Virus titers were determined by the whole egg titration method (26). The titer was calculated by hemagglutination and presented as log EID50 (27).
Chimeric flagellin
Oligonucleotides corresponding to the designated influenza epitopes, i.e. NP335350 (SAAFEDLRVLSFIRGY), NP380393 (ELRSRYWAIRTRSG), and two peptides from the H3 subtype HA, HA91108 (SKAFSNCYPYDVPDYASL) and HA307319 (PKYVKQNTLKLAT), were synthesized in a 380B Applied Biosystems DNA synthesizer with an additional GAT sequence at the 3' of each oligonucleotide in order to preserve the EcoRV restriction site as described elsewhere (8). The synthetic oligonucleotides were inserted at the EcoRV site of the plasmid pLS408 and eventually transformed into a flagellin-negative live vaccine strain of S. dublin SL5928 by transduction, using the phage P22HT105/1 int. Finally the flagella were purified after acidic cleavage and a fine suspension was used for immunization (8).
Immunization and infection of chimeric animals
On the ninth day after PBMC transplantation, human/mouse chimera were immunized once i.n. with a mixture of 25 µg of each hybrid flagellin construct in a total volume of 50 µl PBS or, in the control group, with 75100 µg of the native flagella. This amount was pre-determined as the optimal dose in a preliminary experiment in BALB/c mice. Infection of mice was performed 7 days later by inoculating i.n. the infectious allantoic fluid, 50 µl containing 104 HAU virus per mouse for the sub-lethal dose and 2x102 HAU per mouse for the lethal dose infection. The amount of infectious virus in the challenge stock was 2x1011 EID50. In both immunization and infection, the mice were under a light ether anesthesia. The chimera were sacrificed on day 5 after infection. Their lungs were removed for viral titration.
FACS analysis of donors PBMC and human cell engraftment in chimeric mice
For the evaluation of human cell engraftment in the human/mouse chimera, mice engrafted with human lymphocytes were sacrificed 2729 days after PBMC transplantation. Lymphocytes from lung homogenates as well as peritoneal washes were separated on a Ficoll-Paque gradient (Pharmacia, Uppsala, Sweden) and then incubated for 30 min on ice with a mixture of appropriate fluorescently labeled mAb. After washing, double-fluorescent analysis of human antigens was performed on a FACScan analyzer (Becton Dickinson, Mountain View, CA). The following antibodies were used: anti-CD45phycoerythrin (clone HI30) from PharMingen (San Diego, CA), and anti-CD3PerCP (clone SK7) and anti-CD19FITC (clone 4G7) from Becton Dickinson .
Human Ig determination
Total human Ig was quantified in sera samples by sandwich ELISA using goat F(ab)2-purified anti-human Ig (G + M + A) (Sigma) as the capture agent and peroxidase-conjugated purified goat anti-human Ig (G + M + A) (Sigma) as the detection reagent. Human serum of known Ig concentration was used as the standard. ELISA was performed as described by Marcus et al. (28).
Determination of human Ig specific for influenza
Lung homogenates and sera were tested for specific anti-influenza human antibodies. Native or formalin-fixed virus (100 HAU/ml) was adsorbed to ELISA plates and blocking was performed with 1% BSA in PBS. Rabbit anti-human Ig conjugated to horseradish peroxidase (Sigma) were used as second antibodies. Following the addition of the substrate (ABTS), the plates were read at 414 nm.
Statistical analysis
Statistical analysis was performed using the Stat View II program (Abacus Concepts, Berkeley, CA) on a Macintosh IICi. The F-test was utilized to calculate probability (P) values. Results are presented as mean and SEM of at least two repeated independent experiments, including five to 10 animals per group.
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Results
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Response of the chimeric mice to whole inactivated influenza virus
In order to establish the suitability of the human/mouse radiation chimera for evaluating the synthetic peptide-based vaccine, we have first evaluated their immune response towards inactive purified influenza virus which is known to be protective. The mice were immunized i.p. with 50 µg of the virus on the day of PBMC transplantation, followed by a sub-lethal viral challenge with influenza A/Texas/1/77 14 days post-immunization. The vaccination of human/mouse radiation chimera with the whole killed virus vaccine, without any adjuvant, induced production of specific antibodies. The serum antibody titer was significantly higher (2.4-fold) in the immunized chimera as compared to the control group. Moreover, this vaccination markedly reduced the subsequent virus infection. The lung virus titer after challenge was significantly lower (by 2.7 orders of magnitude) in the immunized chimeras compared to the control mice.
After thus demonstrating the suitability of the human/mouse radiation chimera for evaluating the anti-influenza response following the immunization with inactive influenza virus, we proceeded with the evaluation of the synthetic peptide-based recombinant vaccine designed for humans in this humanized mice model.
FACS analysis of immunized mice for evaluating the engraftment of human PBMC in human/BALB chimera
The successful engraftment of the human cells in the human/mouse chimera was demonstrated in a preliminary experiment showing that a significant proportion of the lymphocytes are of human origin. Yet, up to 2050% of lymphocytes in the peritoneum and up to 4070% of the lung lymphocytes are not human and could be of mouse origin. Figure 1
depicts the pattern of human lung lymphocytes after immunization with the hybrid flagella without further challenge infection. The cells were stained with anti-CD45 antibodies together with anti-CD3 or together with anti-CD19, the staining reagents were shown to be specific to human cellular markers exclusively, and gave negative response with mouse Ig.

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Fig. 1. A typical FACS histogram of human lung lymphocytes in human/mouse radiation chimera, immunized with a mixture of the recombinant flagella (tetra construct) expressing four influenza epitopes. The samples were taken 7 days after the immunization. The cells were separated on a Ficoll gradient and stained with anti-CD45 together with anti-CD3 (A) or together with anti-CD19 (B), conjugated to the respective fluorescence dye. The histograms show that after immunization most of the human cells are T cells (8090%) and almost no B cells (310%) can be detected.
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As shown, most of the human cells are CD3+, i.e. T cells (8090%), and only a minor population is CD19+ (310%). Similar data were obtained for human lymphocytes in the peritoneum. It is of interest that the CD8+/CD4+ ratio in the immunized mice ranged between 1 and 2 as compared to a ratio of 0.30.5 in the untreated chimera. This disproportionate expression of CD8+ cells may suggest that they play a role in the observed protection.
Virus clearance from the lungs following sub-lethal challenge
The mice (six to eight per group in seven repeated experiments) were immunized i.n. 1012 days after the PBMC transplantation, as described in Methods. Ten days later, they were challenged i.n. with 104 HAU in 50 µl allantoic fluid of live A/Texas/1/77 strain of influenza virus. Five days later they were sacrificed and their lungs were removed for virus titration. As shown in Fig. 2
, which depicts the cumulative results, the vaccination with the mixture of the recombinant flagella which includes the four influenza epitopes (abbreviated `tetra construct') enabled the chimera to clear the virus from their lungs significantly more efficiently than the group vaccinated with the native flagella or the group which was not transplanted with PBMC but immunized with the tetra construct. Although the same percentage of human T lymphocytes was detected in both transplanted groups (Fig. 1
), only the mice vaccinated with the hybrid flagellin show the ability to reduce virus burden, indicating specific and efficient local response in the lungs.
Human antibody production in these mice was evaluated both in the serum (before challenge) and in the lungs (after challenge). Immunization with the tetra construct resulted in a significantly higher titer of human antibodies specific for the virus in both serum and lung samples (Fig. 3
). It thus seems that although the proportion of CD19+ lymphocytes as detected by FACS analysis was similarly low in the immunized and control transplanted mice, the production of specific anti-influenza antibody response differs significantly between the two groups.
Survival and weight loss pattern after lethal dose of viral infection
Further to the sub-lethal infection challenge experiment, the ability of the tetra construct preparation to protect the human/mouse chimera from a lethal dose of influenza virus was examined. Figure 4
describes the results of two repeated experiments, and demonstrates the survival of vaccinated and non-vaccinated mice (both transplanted with human PBMC), as well as of another control group that was not transplanted but was vaccinated with the tetra construct. As can be seen, while all control mice that were immunized with the tetra construct but had not been transplanted with the human lymphocytes died within 19 days after the infection, 100% survival was observed in the mice that received the PBMC prior to immunization. This indicates that survival is due to the response of the transplanted human immunocompetent cells. The PBMC by themselves provided a limited beneficial effect, as 50% survival was observed in the control group that was vaccinated with the native flagellin. In Fig. 5
, the body weight loss pattern of the challenged mice is depicted: the transplanted group that was immunized with the tetra flagellin construct shows only a slight reduction in their body weight and a rapid return to normal, while the control group that was transplanted with human PBMC but immunized with the native flagellin lost more weight (the body weight is significantly different between the experimental group and the control groups on days 2233 after transplantation) and the surviving mice started to recover weight only on the last days of the experiment. The non-transplanted, vaccinated control group lost weight rapidly and did not recuperate. The survival of the transplanted group that was immunized with the native flagella is better than that of the non-transplanted group. This can be explained by a memory response of the donor's cells against influenza, due to previous infections. Vaccination with native flagella may also induce a memory response to salmonella antigens in individuals that were previously exposed to salmonella, thus eliciting a non-specific inflammatory reaction (and cytokines secretion) that could also contribute to the protective effect against influenza.
Protection from infection with different strains of influenza
One of the major problems with currently available influenza vaccines is that they are effective only against the strains included in the vaccine. Therefore, it was of interest to examine the ability of the flagellin hybrids that express influenza epitopes to protect mice from different influenza strains that carry various HA and neuraminidase glycoproteins. The B cell epitope that is expressed in the flagellin is conserved in all influenza H3 subtypes, while the T cell epitopes are from regions of the HA and NP highly conserved in other subtypes as well. In the first step, it was shown that rabbit antibodies towards these epitopes can recognize and react in ELISA with different strains of influenza including A/Texas/1/77, A/Aichi/68, A/PR/8/34 and A/Japanese/57. To further test the potential of these epitopes to confer cross-protection in humans, the human/mouse radiation chimera (eight mice per group) were immunized i.n. with the tetra construct. Their resistance to different influenza strain challenge was detected 7 days later and compared to non-transplanted mice that were immunized with the same flagella mixture. The influenza strains that were used for infection were: A/Texas/1/77 (H3N2), A/Japanese/57 (H2N2) and A/PR/8/34 (H1N1). Protective immunity was observed against all three strains, as presented in Fig. 6
. Human Ig specific for each of the above influenza A strains was detected in the sera of all the transplanted and vaccinated mice but not in the control group, as shown in Fig. 7
. No response was detected against influenza B/Harbin/7/94 strain (data not shown). Comparable antibodies response was observed when either fixed or unfixed virus was adsorbed on the ELISA plates (the differences are within the range of the standard variation in the figure). This indicates that most of the antibodies are directed towards the HA and not against the NP epitope, and that more than one epitope of the HA is involved. This is in agreement with the reactivity of the rabbit antisera with the various strains as mentioned above.
It should be noted that the background of the anti-human Ig second antibodies with the immune mouse sera was tested and found to be negative, i.e. no detectable reaction in ELISA.
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Discussion
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This study aimed to establish the ability of human lymphocytes to respond towards peptide-based influenza vaccine. Indeed, the results show that immunization with a mixture of four epitopes from conserved regions in the virus can induce protective and cross-strain immunity. This protection is most probably mediated by both the humoral and the cellular arms of the immune system. This assumption is based on our previous observation that in the normal BALB/c mouse model, immunization with the B cell epitope alone has led to a limited reduction of virus titer in the lungs of the infected mice, whereas an effective and significant protection was achieved when the T cell epitopes were included in the vaccine as well (8). The role of the cellular immunity is further established by the efficient protection of the immunized mice against multiple strains of the virus. Since the sequence of the B cell epitope employed is present only in the H3 HA, the protection against the H1 and H2 strains of influenza is probably mediated by the T cell epitopes that are included in the vaccine. However, the possibility that the protection is mediated by the cross-reacting antibodies to HA 307319 which serves as a Th epitope cannot be excluded, since it was shown that the human antibodies react in ELISA with all the above strains.
The concept of peptide-based vaccines holds several advantages over traditional vaccines, including safety considerations, the relatively long shelf-life, the ability to target the immune response towards specific epitopes that are not suppressive nor hazardous for the host and the possibility of preparing multi-pathogen vaccines. The efficacy of a peptide vaccine is highly dependent on the exact identification of the immunogenic epitopes that confer protection as well as the efficient presentation of these epitopes to the immune system. The idea of using a peptide vaccine for influenza which includes both B and T cells epitopes was previously tested in a mouse model and it has been shown that such a `vaccine' could induce specific local response in the lungs that led to protection of the immunized mice from viral challenge (5,29). In a mouse model, it was shown that the B cell epitope indeed induces high antibody production, while the Th epitope elicited specific lymphocyte proliferation and the CTL epitope was important for cytotoxic activity against infected cells. However, efficient protection was achieved only when the mice were immunized with a mixture of all three epitopes (8).
The purpose of the present study was to adapt this approach for human use. Since the T cell epitopes are MHC restricted, appropriate epitopes had to be selected. Four epitopes were employed: a B cell epitope (HA 91108), the same one shown to be highly efficient in the murine vaccine; a universal Th epitope (HA 307319) that can be bound to many HLA molecules (22); and two CTL epitopes (NP 335350, NP 380393) that are restricted to the most prevalent HLA molecules in the Caucasian population (30,31). Since peptides are usually poor immunogens, the efficacy of a peptide-based vaccine depends on the adequate presentation of the epitopes to the immune system. The influenza epitopes were expressed in the flagellin gene of Salmonella vaccine strain, which provides both carrier and adjuvant function (8). After cleavage of the flagella from the bacteria and the purification steps, the fine suspension of the flagella was used for vaccination. Based on the results from the previous study in mice, in the present experiments all immunizations were performed with a mixture of all four epitopes, in the absence of any adjuvant. To evaluate the capacity of such `vaccine' to stimulate a response of the human immune system, a humanized mouse model has been employed.
The observation of Mosier et al. that human PBMC can be adoptively transferred i.p. into the SCID mouse and that the engrafted cells survive for an extended period of time producing high levels of human Ig has offered many new possibilities in clinical immunology research (reviewed in 32). In particular, many researchers utilized this model for studying the capacity of engrafted lymphocytes to generate primary and secondary human humoral responses (3341), and for viral research studies (4244). Recently, Lubin et al. (10) described a new approach enabling engraftment of human PBMC in normal strains of mice following split-dose lethal irradiation which allows an effective and rapid engraftment of human cells. As previously reported, in such a human/mouse radiation chimera, a marked human humoral as well as cellular (CTL) response could be generated by immunization with either foreign antigens or with allogeneic cells (28,45), rendering this model advantages to the previously used SCID mouse model. A further advantage of this model is that the dissemination of engrafted lymphocytes is very rapid, and both T and B lymphocytes were found by FACS analysis in significant numbers in the lymphoid tissues within a few days post-transplantation (46). NOD/SCID bone marrow has been used since we have previously observed that it leads to better human cell engraftment. Furthermore, the role of xenoreactivity or graft-versus-host disease in this model is minimal (10).
Although the number of human B cells after transplantation is low (Fig. 1
), the chimeric mice are able to produce specific human antibodies in response to i.p. administration of antigens. This is in accord with previous findings showing that towards the second week post-transplantation, the engrafted human B and T cells form follicles in the spleen and lymph nodes. Furthermore, the T cell phenotype was that of memory cells, i.e. mostly CD45RO+ and CD45RA (46). In parallel studies aimed at investigating the cellular immune response of the chimeric mice in detail, the lymphocytes were analyzed by FACS and it was shown that murine lymphocytes appear only ~30 days post-transplantation (47).
In the present study, the human/mouse radiation chimera were immunized with a synthetic peptide-based vaccine against influenza, administered by the i.n. route. This is the first study that reports a contribution of local immunity in the nasal cavity and lungs following i.n. immunization in the human/mouse radiation chimera model. The involvement of local immune responses in the lungs is demonstrated by the presence of specific anti-influenza antibodies in the lung homogenates, by elevation of the proportion of CD8+ lymphocytes in the lungs as well as by the efficient viral clearance from the lungs as a result of immunization with the recombinant flagella. The CD8+ cells cannot lyse infected mouse cells directly because of the differences in MHC molecules on the lymphocytes and on the epithelium of the transplanted mouse.
The mechanism by which the cellular immunity is effective in this system is intriguing, particularly the role of the CTL. CTL restricted to human HLA molecules should not lyse murine cells infected with the virus and hence it is apparently hard to explain the protection based on cellular immunity. However, the controls in which mice that were not transplanted with human cells, but immunized, were not protected, suggest that it is not a murine T cell response from regenerated murine T cells that is responsible for this protection. It is noteworthy that other studies (48) showed that human T cell lines and clones obtained from hu-PBL-SCID chimeras were able to recognize specifically native MHC class II products on murine cells. Yet, there is no evidence that in the system employed in the present study there is a direct lysis of the influenza infected pulmonary cells by the CTL.
It is also possible that the antigen-presenting cells from the donor present viral antigens derived from apoptotic cells and stimulate CTL activity. This phenomena was studied by Albert et al. (49), who showed that dendritic cells could acquire influenza antigens from infected monocytes, process and present them on class I molecules, and eventually stimulate CTL activity. Another and perhaps more likely explanation is that the cellular immunity is cytokine mediated. Indeed, CD8+ and CD4+ cells can secrete cytokines like IFN-
or IL-6 that might clear the infection (5053) even though the T cells would not recognize the influenza antigens directly presented by xenogeneic murine MHC molecules. Furthermore, the ability to release cytokines from Th cells in the model of the human/mouse radiation chimera was shown after in vivo immunization of mice transplanted with human PBMC against hepatitis B virus infection (54).
The flagella mixture could also protect the mice from a lethal dose challenge of the virus, which is the ultimate demonstration of the protective effect. Under these conditions, in which the challenge dose is orders of magnitude higher than that pertaining in natural infection, all the chimeric mice are infected regardless of their immune state. However, whereas none of the immunized mice that had not been transplanted with the human lymphocytes survived the infection and only 50% of those transplanted unimmunized mice survived, the transplanted and immunized group was completely protected and showed 100% survival. The partial protection in the non-vaccinated mice is probably due to polyclonal stimulation and expansion of memory cells originating from the donor. This could be due to either previous exposure of the donor to the antigen or because it is cross-reactive to some extent with other recall antigens, a phenomena that was previously reported for other antigens (28). However, although such partial protection was indeed observed, a significant difference in the efficacy of the recovery process between the immunized and non-immunized groups was observed as evident both by the survival rate and by their weight loss pattern (Figs 4 and 5
). Regrettably, no information is available to us on the HLA phenotypes of the PBMC donors. It is of interest, however, that all of the transplanted mice, recipients of PBMC from different donors, were protected after vaccination with the tetra construct. This may indicate that the epitopes under study are indeed recognized by a wide range of HLA molecules.
One of the most acute problems related to currently existing influenza vaccines is the narrow range of their specificity and their restricted strain-specific activity. The rapid variation in the viral surface glycoproteins leads to the appearance of new strains with high variability in their serospecificity and hence the vaccines containing the outer glycoproteins of some specific strains are limited in their efficacy to these strains. In the present study, we also established the cross-protection capacity of the tetra construct vaccine. All the epitopes that were included in the tetra construct are conserved regions in the respective proteins and, consequently, antibodies against the recombinant flagella could recognize various influenza A strains (but not influenza B). Consequently, immunization of the chimeric mice with the epitopes led to production of specific antibodies and to their protection from sub-lethal dose infection by three different influenza A strains, of H1, H2 or H3 specificity.
Our results demonstrate the ability of a synthetic peptide-based vaccine to confer protection against influenza viral challenge. The recombinant flagellin indeed presents the influenza B and T cell epitopes to the human immune cells in an efficient manner, and induces both humoral and cellular responses. Although the T cell epitopes are restricted to specific HLA molecules, the vaccine was effective in all the experiments in which different donors with unknown HLA typing were utilized. This is an indication for the applicability of this approach for a human vaccine in a heterologous population.
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Acknowledgments
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This paper was written while one of us (R. A) was a scholar-in-residence at the Fogarty International Center for Advanced Study in the Health Sciences, National Institute of Health, Bethesda, MD.
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Abbreviations
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CTL | cytotoxic T lymphocyte |
EID | egg ineffective dose |
HA | hemagglutinin |
HAU | hemagglutination unit |
i.n. | intranasal |
NP | nucleoprotein |
PBMC | peripheral blood mononuclear cell |
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Notes
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Transmitting editor: J. Berzofsky
Received 16 December 1998,
accepted 8 March 1999.
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