Dose dependence of CTL precursor frequency induced by a DNA vaccine and correlation with protective immunity against influenza virus challenge

Intramuscular injection of BALB/c mice with a DNA plasmid encoding nucleoprotein (NP) from influenza virus A/PR/8/34 (H1N1) provides cross-strain protection against lethal challenge with influenza virus A/HK/68 (H3N2). CTL specific for the H-2Kd-restricted epitope NP147-155 are present in these mice and are thought to play a role in the protection. To assess the effectiveness of NP DNA immunization in comparison with influenza virus infection in the induction of CTL responses, we monitored the frequency of CTL precursors (CTLp) in mice following i.m. injection with NP DNA or intranasal infection with influenza virus and showed that the CTLp frequency in NP DNA-immunized mice can reach levels found in mice that had been infected with influenza virus. We also measured the CTLp frequency, anti-NP Ab titers, and T cell proliferative responses in mice that were injected with titrated dosages of NP DNA and documented a correlation of the CTLp frequency and the Ab titers, but not proliferative responses, with the injection dose. Furthermore, we observed a positive correlation between the frequency of NP147-155 epitope-specific CTLp and the extent of protective immunity against cross-strain influenza challenge induced by NP DNA injection. Collectively, these results and our early observations from adoptive transfer experiments of in vitro activated lymphocytes from NP DNA-immunized mice suggest a protective function of NP-specific CTLp in mice against cross-strain influenza virus challenge.     

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats

Conventional influenza vaccines need to be designed and manufactured yearly. However, they occasionally provide poor protection owing to antigenic mismatch. Hence, there is an urgent need to develop universal vaccines against influenza virus. Using nucleoprotein(NP) and extracellular domain of matrix protein 2(M2e) genes from the influenza A virus A/Beijing/30/95(H3N2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders. The recombinant vaccinia viruses were used to immunize BALB/C mice. Humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34(PR8). NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP, while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e. All recombinant viruses elicited NP-and M2e-specific cellular immune responses in mice. Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e. Furthermore, RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD_(50) of PR8. Therefore, the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP, which expresses a fusion antigen of full-length NP preceded by four M2e repeats. These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses, and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.     

Influenza A virus nucleoprotein derived from Escherichia coli or recombinant vaccinia (Tiantan) virus elicits robust cross-protection in mice

Immunity to conserved viral antigens is an attractive approach to develop a universal vaccine against epidemic and pandemic influenza. A nucleoprotein (NP)-based vaccine has been explored and preliminary studies have shown promise. However, no study has explored the immunity and cross-protective efficacy of recombinant NP derived from Escherichia coli compared with recombinant vaccinia virus (Tiantan). Recombinant NP protein (rNP) from influenza virus A/Jingke/30/95(H3N2) was obtained from E. coli and recombinant vaccinia virus (Tiantan) RVJ1175NP. Purified rNP without adjuvant and RVJ1175NP were used to immunize BALB/c mice intramuscularly. Humoral immune responses were detected by ELISA, while cell-mediated immune responses were measured by ex vivo IFN-γ ELISPOT and in vivo cytotoxicity assays. The cross-protective efficacy was assessed by a challenge with a heterosubtype of influenza virus A/PR/8/34(H1N1). Our results demonstrate that a high dose (90 μg) of rNP induced NP-specific antibodies and T cell responses that were comparable with those of RVJ1175NP in mice. Importantly, the survival ratio (36, 73, and 78%) of the vaccinated mice after the influenza virus A/PR/8/34(H1N1) challenge was rNP vaccine dose-dependent (10, 30, and 90 μg, respectively), and no significant differences were observed between the rNP- and RVJ1175NP-immunized (91%) mice. Influenza A virus NP derived from E. coli or recombinant vaccinia (Tiantan) virus elicited cross-protection against influenza virus in mice, and the immune response and protective efficacy of rNP were comparable to RVJ1175NP. These data provide a basis for the use of prokaryotically expressed NP as a candidate universal influenza vaccine.     

Mucosal and systemic immune responses to a human immunodeficiency virus type 1 epitope induced upon vaginal infection with a recombinant influenza A virus

The humoral and cellular immune responses in the genital mucosa likely play an important role in the prevention of sexually transmitted infections, including infection with human immunodeficiency virus type 1 (HIV-1). Here we show that vaginal infection of progesterone-treated BALB/c mice with a recombinant influenza virus bearing the immunodominant P18IIIB cytotoxic T-lymphocyte (CTL) epitope of the gp160 envelope protein from an HIV-1 IIIB isolate (P18IIIB; RIQRGPGRAFVTIGK) can induce a specific immune response in regional mucosal lymph nodes, as well as in a systemic site (the spleen). A single inoculation of mice with the recombinant influenza virus induced long-lasting (at least 5 months) antigen-specific CTL memory detectable as a rapid recall of effector CTLs upon vaginal infection with recombinant vaccinia virus expressing HIV-1 IIIB envelope gene products. Long-term antigen-specific CTL memory was also induced and maintained in distant mucosal tissues when mice were intranasally immunized with the recombinant influenza virus. These results indicate that mucosal immunization and, in particular, local vaginal immunization with recombinant influenza virus can provide strong, durable immune responses in the female genital tract of mice.     

甲型流感病毒核壳蛋白在BALB／c小鼠中酶联免疫斑点法表位的筛选及其与CTL表位的相关性研究

目的：利用甲型流感病毒A／Johannesburg／33／94（H3N2）核壳蛋白（NP）全长肽库筛选BAI卫／c（H-2^d）小鼠中NP酶联免疫斑点法（EUSPOT）表位,研究其和细胞毒性T淋巴细胞（CTL）表位的一致性关系,为使用ELlSPOT评价流感病毒NP疫苗的细胞免疫效果提供实验依据。方法：以甲型流感病毒A／PR／8／34（H1N1）（PR8）感染BALB／c（H-2^d）小鼠后,通过检测T细胞分泌γ-干扰素（IFN-1）的ELISPOT法和体内CTL法检测NP所诱发的细胞免疫反应,综合分析ELlSPOT和CTL表位肽之间的关系。结果：Pep36（NP第141-155位氨基酸残基,SNLNDTTYQRTRALV）和Pep37（NP第145-159位氨基酸残基,DTTYQRTRALVRTGM）可以诱发较强的ELlSPOT反应,根据Pep36和Pep37共有序列合成的Pep147-155（NP第147-155位氨基酸残基,TYQRTRALV）可以诱发与这2条多肽相同强度的ELlSPOT反应,表明Pep147-155为NP诱发ELlSPOT反应的最强表位,体内CTL也表明它是最强的CTL表位;Pep95（NP第377-391位氨基酸残基,STLELRSRYWAIRTR）、Pep96（NP第381-395位氨基酸残基,LRSRYWAIRTRSGGN）和其他表位肽诱发的ELISPOT反应较弱,体内CTL反应也较弱。结论：BALB／c（H-2^d）小鼠中,甲型流感病毒NP诱发ELlSPOT反应和CTL反应的表位肽高度相关;实验结果为使用ELlSPOT评价流感病毒NP疫苗的细胞免疫效果提供了实验依据。     

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.     

Dendritic cells recruitment and in vivo priming of CD8+ CTL induced by a single topical or transepithelial immunization via the buccal mucosa with measles virus nucleoprotein.

The buccal mucosa, a prototype of pluristratified mucosal epithelia, contains a network of directly accessible class II(+) epithelial dendritic cells (DC), similar to skin Langerhans cells. We showed that a single buccal immunization with measles virus nucleoprotein (NP), by either topical application onto or intradermal injection in the buccal mucosa, induced in vivo priming of protective class I-restricted specific CD8(+) CTL. Both routes of immunization with NP induced a rapid recruitment of DC into the mucosa, which peaked at 2 h and decreased by 24 h. Treatment of mice with Flt3 ligand resulted in an increased number of DC in the buccal mucosa and enhanced the frequency of IFN-gamma-producing NP-specific effectors and the NP-specific CTL response generated after buccal immunization with NP. Finally, NP-pulsed bone marrow-derived DC induced NP-specific IFN-gamma-producing cells upon adoptive transfer to naive mice. These data demonstrate that a viral protein delivered to DC of the buccal mucosa induces in vivo priming of protective anti-viral CD8(+) CTL.     

Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein

Cytotoxic T lymphocytes (CTLs) are proposed to be critical for protection from intracellular pathogens such as Ebola virus. However, there have been no demonstrations that protection against Ebola virus is mediated by Ebola virus-specific CTLs. Here, we report that C57BL/6 mice vaccinated with Venezuelan equine encephalitis virus replicons encoding the Ebola virus nucleoprotein (NP) survived lethal challenge with Ebola virus. Vaccination induced both antibodies to the NP and a major histocompatibility complex class I-restricted CTL response to an 11-amino-acid sequence in the amino-terminal portion of the Ebola virus NP. Passive transfer of polyclonal NP-specific antiserum did not protect recipient mice. In contrast, adoptive transfer of CTLs specific for the Ebola virus NP protected unvaccinated mice from lethal Ebola virus challenge. The protective CTLs were CD8(+), restricted to the D(b) class I molecule, and recognized an epitope within amino acids 43 to 53 (VYQVNNLEEIC) in the Ebola virus NP. The demonstration that CTLs can prevent lethal Ebola virus infection affects vaccine development in that protective cellular immune responses may be required for optimal protection from Ebola virus.     

Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice

A recombinant baculovirus expressing the hemagglutinin gene of the influenza virus, A/PR/8/34 (H1N1), under the control of the chicken beta-actin promoter, was constructed. To determine the induction of protective immunity in vivo, mice were inoculated with the recombinant baculovirus by intramuscular, intradermal, i.p., and intranasal routes and then were challenged with a lethal dose of the influenza virus. Intramuscular or i.p. immunization with the recombinant baculovirus elicited higher titers of antihemagglutinin Ab than intradermal or intranasal administration. However, protection from a lethal challenge of the influenza virus was only achieved by intranasal immunization of the recombinant baculovirus. Surprisingly, sufficient protection from the lethal influenza challenge was also observed in mice inoculated intranasally with a wild-type baculovirus, as evaluated by reductions in the virus titer, inflammatory cytokine production, and pulmonary consolidations. These results indicate that intranasal inoculation with a wild-type baculovirus induces a strong innate immune response, which protects mice from a lethal challenge of influenza virus.     

Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization.

DNA immunization offers a novel means to induce cellular immunity in a population with a heterogeneous genetic background. An immunorecessive cytotoxic T-lymphocyte (CTL) epitope in influenza virus nucleoprotein (NP), residues 218 to 226, was identified when mice were immunized with a plasmid DNA encoding a full-length mutant NP in which the anchor residues for the immunodominant NP147-155 epitope were altered. Mice immunized with wild-type or mutant NP DNA were protected from lethal cross-strain virus challenge, and the protection could be adoptively transferred by immune splenocytes, indicating the role of cell-mediated immunity in the protection. DNA immunization is capable of eliciting protective cellular immunity against both immunodominant and immunorecessive CTL epitopes in the hierarchy seen with virus infection.     

Induction by DNA immunization of a protective antitumor cytotoxic T lymphocyte response against a minimal-epitope-expressing tumor

 In order to examine the use of DNA immunization to block tumor growth, we have developed a model system in which a defined 9-amino-acid epitope from the nucleoprotein of influenza virus is used as a surrogate tumor-associated antigen. A mastocytoma cell line of DBA/2 origin (P815) was transfected with a plasmid encoding the minimal H-2K^d-restricted NP(147–155) cytotoxic T lymphocyte (CTL) epitope, pCMV/NPep, to generate the cell line designated P815-NPep. Mice primed and boosted once with a plasmid encoding the full-length NP gene, pCMV/NP, but not with the minigene pCMV/NPep, developed a strong NP(147–155)-specific CTL response within 2 weeks after the boost. When challenged with 10⁴ P815-NPep cells, pCMV/NP-immunized DBA/2 mice were protected from tumor challenge, whereas control mice immunized with the vector backbone rapidly developed lethal tumor. Importantly, the P815-NPep-immune mice were also protected from a subsequent challenge with the untransfected parental tumor P815. By depleting the NP-immune mice of either CD4⁺ or CD8⁺ T cells and then challenging with 10⁴ P815-NPep tumor cells, it was determined that the CD8-depleted mice rapidly developed tumors, whereas the CD4-depleted or non-treated mice were protected. These data clearly indicate that intramuscular (i.m.) plasmid DNA immunization can be used to mobilize an effective CD8⁺ CTL-mediated antitumor response. Received: 8 May 1997 / Accepted: 28 August 1997     

The cytolytic activity of pulmonary CD8+ lymphocytes, induced by infection with a vaccinia virus recombinant expressing the M2 protein of respiratory syncytial virus (RSV), correlates with resistance to RSV infection in mice.

Previous studies demonstrated that the pulmonary resistance to respiratory syncytial virus (RSV) challenge induced by immunization with a recombinant vaccinia virus expressing the M2 protein of RSV (vac-M2) was significantly greater 9 days after immunization than at 28 days and was mediated predominantly by CD8+ T cells. In this study, we have extended these findings and sought to determine whether the level of CD8+ cytotoxic T-lymphocyte (CTL) activity measured in vitro correlates with the resistance to RSV challenge in vivo. Three lines of evidence documented an association between the presence of pulmonary CTL activity and resistance to RSV challenge. First, vac-M2 immunization induced pulmonary CD8+ CTL activity and pulmonary resistance to RSV infection in BALB/c (H-2d) mice, whereas significant levels of pulmonary CTL activity and resistance to RSV infection were not seen in BALB.K (H-2k) or BALB.B (H-2b) mice. Second, pulmonary CD8+ CTL activity was not induced by infection with other vaccinia virus-RSV recombinants that did not induce resistance to RSV challenge. Third, the peak of pulmonary CTL activity correlated with the peak of resistance to RSV replication (day 6), with little resistance being observed 45 days after immunization. An accelerated clearance of virus was not observed when mice were challenged with RSV 45 days after immunization with vac-M2. The results indicate that resistance to RSV induced by immunization with vac-M2 is mainly mediated by primary pulmonary CTLs and that this resistance decreases to very low levels within 2 months following immunization. The implications for inclusion of CTL epitopes into RSV vaccines are discussed in the context of these observations.     

Cytotoxic T-Lymphocyte Epitope Immunodominance in the Control of Choroid Plexus Tumors in Simian Virus 40 Large T Antigen Transgenic Mice

The simian virus 40 (SV40) large tumor antigen (Tag) is a virus-encoded oncoprotein which is the target of a strong cytotoxic T-lymphocyte (CTL) response. Three immunodominant H-2(b)-restricted epitopes, designated epitopes I, II/III, and IV, have been defined. We investigated whether induction of CTLs directed against these Tag epitopes might control Tag-induced tumors in SV11(+) (H-2(b)) mice. SV11(+) mice develop spontaneous tumors of the choroid plexus due to expression of SV40 Tag as a transgene. We demonstrate that SV11(+) mice are functionally tolerant to the immunodominant Tag CTL epitopes. CTLs specific for the H-2Kb-restricted Tag epitope IV were induced in SV11(+) mice following adoptive transfer with unprimed C57BL/6 spleen cells and immunization with recombinant vaccinia viruses expressing either full-length Tag or the H-2Kb-restricted epitope IV as a minigene. In addition, irradiation of SV11(+) mice prior to adoptive transfer with unprimed C57BL/6 spleen cells led to the priming of epitope IV-specific CTLs by the endogenous Tag. Induction of epitope IV-specific CTLs in SV11(+) mice by either approach correlated with increased life span and control of the choroid plexus tumor progression, indicating that CTLs specific for the immunodominant Tag epitope IV control the progressive growth of spontaneous tumors induced by this DNA virus oncogene in transgenic mice.     

Virus-like particle vaccine induces protective immunity against homologous and heterologous strains of influenza virus

Recurrent outbreaks of highly pathogenic avian influenza virus pose the threat of pandemic spread of lethal disease and make it a priority to develop safe and effective vaccines. Influenza virus-like particles (VLPs) have been suggested to be a promising vaccine approach. However, VLP-induced immune responses, and their roles in inducing memory immune responses and cross-protective immunity have not been investigated. In this study, we developed VLPs containing influenza virus A/PR8/34 (H1N1) hemagglutinin (HA) and matrix (M1) proteins and investigated their immunogenicity, long-term cross-protective efficacy, and effects on lung proinflammatory cytokines in mice. Intranasal immunization with VLPs containing HA induced high serum and mucosal antibody titers and neutralizing activity against PR8 and A/WSN/33 (H1N1) viruses. Mice immunized with VLPs containing HA showed little or no proinflammatory lung cytokines and were protected from a lethal challenge with mouse-adapted PR8 or WSN viruses even 5 months postimmunization. Influenza VLPs induced mucosal immunoglobulin G and cellular immune responses, which were reactivated rapidly upon virus challenge. Long-lived antibody-secreting cells were detected in the bone marrow of immunized mice. Immune sera administered intranasally were able to confer 100% protection from a lethal challenge with PR8 or WSN, which provides further evidence that anti-HA antibodies are primarily responsible for preventing infection. Taken together, these results indicate that nonreplicating influenza VLPs represent a promising strategy for the development of a safe and effective vaccine to control the spread of lethal influenza viruses.     

Induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand RNA virus vectors

A recombinant Newcastle disease virus (rNDV) expressing simian immunodeficiency virus (SIV) Gag protein (rNDV/SIVgag) was generated. The rNDV/SIVgag virus induced Gag-specific cellular immune responses in mice, leading to a specific anti-Gag antiviral immunity. This was evidenced by the inhibition of growth of recombinant vaccinia virus expressing an identical Gag antigen (rVac/SIVgag) but not of wild-type vaccinia virus in rNDV/SIVgag-immunized mice. Among intravenous, intraperitoneal, or intranasal immunization routes, intranasal administration induced the strongest protective response against challenge with rVac/SIVgag. We further demonstrated that these immune responses were greatly enhanced after booster immunization with recombinant influenza viruses expressing immunogenic portions of SIV Gag. The magnitude of the protective immune response correlated with the levels of cellular immune responses to Gag, which were still evident 9 weeks after immunization. These results suggest that rNDV and influenza virus vectors are suitable candidate vaccines against AIDS as well as against other infectious diseases.     

Characterization of two distinct major histocompatibility complex class I Kk-restricted T-cell epitopes within the influenza A/PR/8/34 virus hemagglutinin.

Cytotoxic T-lymphocyte (CTL) clones specific for the influenza A/PR/8/34 virus hemagglutinin (HA) were isolated by priming CBA mice with a recombinant vaccinia virus expressing the HA molecule. The epitopes recognized by two of these clones, which were CD8+, Kk restricted, and HA subtype specific, were defined by using a combination of recombinant vaccinia viruses expressing HA fragments and synthetic peptides. One epitope is in the HA1 subunit at residues 259 to 266 (numbering from the initiator methionine), amino acid sequence FEANGNLI, and the other epitope is in the HA2 subunit at residues 10 to 18 (numbering from the amino terminus of the HA2 subunit), sequence IEGGWTGMI. These two peptides are good candidates for naturally processed HA epitopes presented during influenza infection, as they are the same length (eight and nine residues) as other naturally processed viral peptides presented to CTL. A comparison of the sequences of these two new epitopes with those of the three previously published Kk-restricted T-cell epitopes showed some homology among all of the epitopes, suggesting a binding motif. In particular, an isoleucine residue at the carboxy-terminal end is present in all of the epitopes. On the basis of this homology, we predicted that the Kk-restricted epitope in influenza virus nucleoprotein, previously defined as residues 50 to 63, was contained within residues 50 to 57, sequence SDYEGRLI. This shorter peptide was found to sensitize target cells at a 200-fold lower concentration than did nucleoprotein residues 50 to 63 when tested with a CTL clone, confirming the alignment of Kk-restricted epitopes.     

Cytotoxic T cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse L cells

L cells expressing either the A/NT/60/68 nucleoprotein or the A/PR/8/34 (H1) hemagglutinin by DNA mediated gene transfer were used to investigate recognition by influenza A specific cytotoxic T lymphocytes (CTL). A subpopulation of CTL that recognized the H1 hemagglutinin was detected in mice primed with either A/PR/8/34 (H1N1) or A/JAP/305/57 (H2N2) influenza viruses. However, neither CTL from mice primed with A/NT/60/68 (H3N2) nor the recombinant virus X31 (H3N2) showed any activity on L cells expressing H1. These results showed that the majority of fully crossreactive CTL do not recognize the hemagglutinin molecule. A comparison between nucleoprotein and hemagglutinin transfected L cells reveals the nucleoprotein as the major target for CTL that are crossreactive on the three pandemic strains of human influenza A virus.     

Selection of the same major T cell determinants of influenza nucleoprotein after vaccination or exposure to infectious virus.

In the present study, we have compared the T cell antigenic determinants on nucleoprotein (NP) of influenza A/NT/60/68 virus recognized by BALB/c mice (H-2d) after vaccination using several different vehicles with the determinants recognized after exposure to infectious virus. Mice were immunized s.c. with: 1) purified recombinant NP with three different adjuvants--alum, saponin, or CFA; 2) whole inactivated A/Okuda virus in PBS or saponin; or 3) live attenuated Salmonella typhimurium AroA- vector expressing NP. A series of overlapping synthetic peptides that cover more than 90% of the amino acid sequence of NP were used to map the Th cell epitopes. The results showed that the same limited number of major epitopes were recognized after each of the different immunization regimes. Secondary in vivo boosting using the same vehicles as for the primary immunization did not increase the number of different T cell sites recognized. The T cell responses after intranasal infection with infectious A/NT/60/68 or A/PR/8/34 virus also showed a similar pattern of recognition of the major CD4-positive T cell epitopes. The only exception was that the region corresponding to residues 401-419 was only recognized after exposure to NP from A/NT/60/68 but not A/PR/8/34. This is probably because the two viruses differ in amino acid sequence at positions 408 and 411 within this part of the NP molecule. In contrast to the results observed with CD4-positive T cell epitopes, the major determinant recognized by CD8-positive T cells was only presented after live viral infection. The results in this study have important implications for vaccine design, inasmuch as they indicate that the same dominant CD4 T cell determinants on NP presented by vaccination with NP are also recognized by T cells from mice exposed to infectious virus.     

Activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs) by human dendritic cells infected with an attenuated influenza A virus expressing a CTL epitope derived from the HER-2/neu proto-oncogene

The development of cancer vaccines requires approaches to induce expansion and functional differentiation of tumor antigen-specific cytotoxic T lymphocyte (CTL) effectors which posses cytolytic capability and produce cytokines. Efficient induction of such cells is hindered by the poor immunogenicity of tumor antigens and by the poor transduction efficiency of dendritic cells (DCs) with current nonreplicating vectors. We have investigated the use of influenza A virus, a potent viral inducer of CTLs, as a vector expressing the immunodominant HER-2 CTL epitope KIF (E75). For this purpose, an attenuated influenza A/PR8/34 virus with a truncated nonstructural (NS1) gene was generated containing the E75 epitope in its neuraminidase protein (KIF-NS virus). Stimulation of peripheral blood mononuclear cells from healthy donors and of tumor-associated lymphocytes from ovarian and breast cancer patients with DCs infected with KIF-NS virus (KIF-NS DC) induced CTLs that specifically recognized the peptide KIF and HER-2-expressing tumors in cytotoxicity assays and secreted gamma interferon (IFN-gamma) and interleukin-2 at recall with peptide. Priming with KIF-NS DCs increased the number of E75(+) CD45RO(+) cells by more than 10-fold compared to nonstimulated cells. In addition, KIF-NS virus induced high levels of IFN-alpha in DCs. This is the first report demonstrating induction of human epitope-specific CTLs against a tumor-associated antigen with a live attenuated recombinant influenza virus vector. Such vectors may provide a novel approach for tumor antigen delivery, lymphocyte activation, and differentiation in human cancer vaccine development.     

Rectification of age-associated deficiency in cytotoxic T cell response to influenza A virus by immunization with immune complexes

Decline in cellular immunity in aging compromises protection against infectious diseases and leads to the increased susceptibility of the elderly to infection. In particular, Ag-specific cytotoxic T lymphocyte (CTL) response against virus is markedly reduced in an aged immune system. It is of great importance to explore novel strategy in eliciting effective antiviral CTL activity in the elderly. In this study, the efficacy and mechanisms of immunization with immune complexes in overcoming age-associated deficiency in cellular immunity were investigated. In this study, we show that the severely depressed CTL response to influenza A in aged mice can be significantly restored by immunization with immune complexes consisting of influenza A virus and mAb to influenza A nucleoprotein. The main mechanisms underlying this recovery of CTL response induced by immune complex immunization in aged mice are enhanced dendritic cell function and elevated production of IFN-gamma in both CD4(+) Th1 and CD8(+) CTLs. Thus, these results demonstrate that immune complex immunization may represent a novel strategy to elicit effective virus-specific cytotoxic response in an aged immune system, and possibly, to overcome age-related immune deficiency in general.