Recombinant herpes simplex virus type 1 (HSV-1) codelivering interleukin-12p35 as a molecular adjuvant enhances the protective immune response against ocular HSV-1 challenge

An important aspect of ocular herpes simplex virus type 1 (HSV-1) vaccine development is identification of an appropriate adjuvant capable of significantly reducing both virus replication in the eye and explant reactivation in trigeminal ganglia. We showed recently that a recombinant HSV-1 vaccine expressing interleukin-4 (IL-4) is more efficacious against ocular HSV-1 challenge than recombinant viruses expressing IL-2 or gamma interferon (IFN-gamma) (Y. Osorio and H. Ghiasi, J. Virol. 77:5774-5783, 2003). We have now constructed and compared recombinant HSV-1 viruses expressing IL-12p35 or IL-12p40 molecule with IL-4-expressing HSV-1 recombinant virus. BALB/c mice were immunized intraperitoneally with IL-12p35-, IL-12p40-, IL-12p35+IL-12p40-, or IL-4-expressing recombinant HSV-1 viruses. Controls included mice immunized with parental virus and mice immunized with the avirulent strain KOS. The efficacy of each vaccine in protecting against ocular challenge with HSV-1 was assessed in terms of survival, eye disease, virus replication in the eye, and explant reactivation. Neutralizing antibody titers, T-cell responses, and expression of 32 cytokines and chemokines were also evaluated. Mice immunized with recombinant HSV-1 expressing IL-12p35 exhibited the lowest virus replication in the eye, the most rapid virus clearance, and the lowest level of explant reactivation. The higher efficacy against ocular virus replication and explant reactivation correlated with higher neutralizing antibody titers, cytotoxic-T-lymphocyte activities, and IFN-gamma expression in recombinant HSV-1 expressing IL-12p35 compared to other vaccines. Mice immunized with both IL-12p35 and IL-12p40 had lower neutralizing antibody responses than mice immunized with IL-12p35 alone. Our results confirm that recombinant virus vaccines expressing cytokine genes can enhance the overall protection against infection, with the IL-12p35 vaccine being the most efficacious of those tested. Collectively, the results support the potential use of IL-12p35 as a vaccine adjuvant, without the toxicity-associated concerns of IL-12.     

Immune response and cytokine production following immunization with experimental herpes simplex virus 1 (HSV-1) vaccines

Balb/c mice were immunized with the recombinant fusion protein gD1/313 (FpgD1/313 representing the ectodomain of HSV-1 gD), with the non-pathogenic ANGpath gE-del virus, with the plasmid pcDNA3.1-gD expressing full-length gD1 and with the recombinant immediate early (IE) HSV-1 protein ICP27. Specific antibodies against these antigens (as detected by ELISA) reached high titers with the exception of the DNA vaccine. High-grade protection against challenge with the virulent strain SC16 was found following immunization with the pcDNA3.1-gD plasmid and with the gE-del virus. Medium grade, but satisfactory protection developed after immunization with the FpgD1/313 and minimum grade protection was seen upon immunization with the IE/ICP27 polypeptide. A considerable response of peripheral blood cells (PBL) and splenocytes in the lymphocyte transformation test (LTT) was found in mice immunized with FpgD1/313, with the pcDNA3.1-gD plasmid and with the live ANGpathgE-del virus. For lymphocyte stimulation in vitro, the FpgD1/313 antigen was less effective than the purified gD1/313 polypeptide (cleaved off from the fusion protein); both proteins elicited higher proliferation at the 5 mug per 0.1 mL dose than at the 1 mug per 0.1 mL dose. The secretion of Th type 1 (TNF, IFN-gamma and IL-2) and Th type 2 (IL-4 and IL-6) cytokines was tested in the medium fluid of purified PBL and splenocyte cultures; their absolute values were expressed in relative indexes. The PBL from FpgD1/313 immunized mice showed increased secretion of both T(H)1 (TNF) as well as T(H)2 (IL-4) cytokines (7-10-fold, respectively). Splenocytes from FpgD1/313 immunized mice showed a significant (23-fold) increase in IL-4 production.     

Yellow fever virus encephalitis: properties of the brain-associated T-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for CD4+ lymphocytes

Viral encephalitis caused by neuroadapted yellow fever 17D virus (PYF) was studied in parental and gamma interferon (IFN-gamma)-deficient (IFN-gamma knockout [GKO]) C57BL/6 mice. The T-cell responses which enter the brain during acute fatal encephalitis of nonimmunized mice, as well as nonfatal encephalitis of immunized mice, were characterized for relative proportions of CD4+ and CD8+ cells, their proliferative responses, and antigen-specific expression of cytokines during stimulation in vitro. Unimmunized mice accumulated only low levels of T cells within the brain during fatal disease, whereas the brains of immunized mice contained higher levels of both T-cell subsets in response to challenge, with CD8+ cells increased relative to the CD4+ subset. The presence of T cells correlated with the time at which virus was cleared from the central nervous system in both parental and GKO mice. Lymphocytes isolated from the brains of challenged immunized mice failed to proliferate in vitro in response to T-cell mitogens or viral antigens; however, IFN-gamma, interleukin 4 (IL-4), and, to a lesser extent, IL-2 were detectable after stimulation. The levels of IFN-gamma, but not IL-2 or IL-4, were augmented in response to viral antigen, and this specificity was detectable in the CD4+ compartment. When tested for the ability to survive both immunization and challenge with PYF virus, GKO and CD8 knockout mice did not differ from parental mice (80 to 85% survival), although GKO mice exhibited a defect in virus clearance. In contrast, CD4 knockout and Igh-6 mice were unable to resist challenge. The data implicate antibody in conjunction with CD4+ lymphocytes bearing a Th1 phenotype as the critical factors involved in virus clearance in this model.     

Efficacy of recombinant herpes simplex virus 1 glycoprotein D candidate vaccines in mice

To compare the immunogenity of the herpes simplex virus 1 (HSV-1/HHV-1) recombinant glycoprotein D (gD1), as a potential protective vaccine, Balb/c mice were immunized with either gD1/313 (the ectodomain of the gD1 fusion protein consisting of 313 amino acid residues), or the plasmid pcDNA3.1-gD (coding for a full length gD1 protein, FLgD1). A live attenuated HSV-1 (deleted in the gE gene), and a HSV-1 (strain HSZP)-infected cell extract served as positive controls, and three non-structural recombinant HSV-1 fusion proteins (ICP27, UL9/OBP and thymidine kinase--TK) were used as presumed non-protective (negative) controls. Protection tests showed that the LD50 value of the challenging infectious virus increased 90-fold in mice immunized with ICP27, but remained unchanged in other control mice immunized with TK and OBP polypeptides. A significant protection (the LD50 value of challenging virus increased 800-fold) was noted following immunization with gD1/313, while immunization with the gE-del virus and/or the gD1 DNA vaccine resulted in a more than 4,000-fold increase of the challenging virus dose killing 50% of the animals. Using ELISA, elevated antibody titers were detected following immunizations with gD1/313, gE-del virus, and/or HSV-1-infected-cell extract. In addition, all of the three non-structural proteins elicited a good humoral response (with titres ranging from 1:16,000 to 1:128,000). The lowest IgG response (1:8,000) was noted after immunization with the gD1 DNA vaccine. Peripheral blood leukocytes (PBLs) as well as splenocytes from mice immunized with gD1/313, gE-del virus, and gD1-plasmid responded in lymphocyte transformation test (LTT) to the presence of purified gD1/313 antigen. For PBLs, the most significant stimulation of thymidine incorporation was registered at a gD1/313 concentration of 5 microg/100 microl, while the splenocytes from DNA vaccine-immunized mice responded already at a concentration of 1 microg/100 microl.     

4-1BBL enhances anti-tumor responses in the presence or absence of CD28 but CD28 is required for protective immunity against parental tumors

A20 is an aggressive BALB/c B cell lymphoma that, despite its expression of B7-2, rapidly forms tumors in syngeneic mice. We have generated A20 transfectants expressing elevated levels of B7-2 (A20/B7-2high) or 4-1BBL (A20/4-1BBL(low,mod,high)) and found that mice which were able to reject the A20/B7-2 or A20/4-1BBL transfectants were also resistant to subsequent systemic challenge with the parental cell line. To assess whether the effectiveness of 4-1BBL in enhancing anti-tumor immunogenicity was dependent on additional signals from B7-CD28 interaction, we injected the A20 variants into BALB/c CD28(-/-) mice. We found that CD28(-/-) mice were able to reject the A20/4-1BBL variants while A20/B7-2 cells formed tumors. However, when the A20/4-1BBL resistant CD28(-/-) mice were systemically challenged with the A20 parental line, tumors formed rapidly. Upon restimulation in vitro, splenocytes from A20/4-1BBL immunized CD28(+/+) mice were able to kill parental tumors whereas splenocytes from CD28(-/-) mice showed a reduction in CTL activity against A20 or A20/4-1BBL targets. Examination of cytokine production by the immunized animals indicated that the CD28(-/-) splenocytes secreted substantially less IL-2 as well as reduced levels of IFN-gamma compared with their CD28(+/+) counterparts. Thus, 4-1BBL expressing tumors are capable of priming CTL responses against 4-1BBL transfected as well as parental tumors in the absence of CD28. However, in the absence of CD28 signaling, the production of cytokines and particularly IL-2 was lower, resulting in a weaker CTL recall response and reduced ability to survive challenge with parental tumor.     

Immunization with replication-defective mutants of herpes simplex virus type 1: sites of immune intervention in pathogenesis of challenge virus infection.

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) were used as a new means to immunize mice against HSV-1-mediated ocular infection and disease. The effects of the induced immune responses on pathogenesis of acute and latent infection by challenge virus were investigated after corneal inoculation of immunized mice with virulent HSV-1. A single subcutaneous injection of replication-defective mutant virus protected mice against development of encephalitis and keratitis. Replication of the challenge virus at the initial site of infection was lower in mice immunized with attenuated, wild-type parental virus (KOS1.1) or replication-defective mutant virus than in mice immunized with uninfected cell extract or UV-inactivated wild-type virus. Significantly, latent infection in the trigeminal ganglia was reduced in mice given one immunization with replication-defective mutant virus and was completely prevented by two immunizations. Acute replication in the trigeminal ganglia was also prevented in mice immunized twice with wild-type or mutant virus. The level of protection against infection and disease generated by immunization with replication-defective mutant viruses was comparable to that of infectious wild-type virus in all cases. In addition, T-cell proliferative and neutralizing antibody responses following immunization and corneal challenge were of similar strength in mice immunized with replication-defective mutant viruses or with wild-type virus. Thus, protein expression by forms of HSV-1 capable of only partially completing the replication cycle can induce an immune response in mice that efficiently decreases primary replication of virulent challenge virus, interferes with acute and latent infection of the nervous system, and inhibits the development of both keratitis and systemic neurologic disease.     

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.     

The adjuvant effects of co-stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination

Because DNA vaccines on their own tend to induce weak immune responses in humans, adjuvant methods are needed in order to improve their efficacy. The co-stimulatory molecules 4-1BBL, OX40L, and CD70 have been shown to induce strong T cell activities; therefore, in this study, we investigated whether they may be used as molecular adjuvants for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcDS2) in eliciting strong cellular and memory responses. Compared to mice immunized with pcDS2 alone, addition of the co-stimulatory molecules increased T cell proliferation and an HBsAg-specific antibody response that was marked with a higher ratio of IgG2a/IgG1. Importantly, pcDS2 plus these co-stimulatory molecules elicited a higher level of IFN-gamma and IL-4 in CD4(+) T cells and a higher level of IFN-gamma in CD8(+) T cells. In addition, a significantly robust antigen-specific cytotoxic T lymphocyte (CTL) response and the production of long-term memory CD8(+) T cells were also observed in the groups immunized with pcDS2 plus 4-1BBL, OX40L, or CD70. Consistently, as late as 100 days after immunization, upregulated expressions of BCL-2, Spi2A, IL-7Ra, and IL-15Ra were still observed in mice immunized with pcDS2 plus these co-stimulatory molecules, suggesting the generation of memory T cells in these groups. Together, these results suggest that the co-stimulatory molecules 4-1BBL, OX40L, or CD70 can enhance the immunogenicity of HBsAg DNA vaccines, resulting in strong humoral, cellular, and memory responses. This approach may lead to an effective therapeutic vaccine for chronic hepatitis B virus (HBV) infection.     

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.     

TH(1)- and TH(2)-TYPE cytokine expression by activated t lymphocytes from the lung and spleen during the inflammatory response to respiratory syncytial virus

RSV is an important cause of lower respiratory tract illness in infants and the elderly worldwide. The components involved in immunity and those that contribute to inflammation of RSV-induced disease are not clearly understood. To address the relationship between activation antigen and cytokine expression, intracellular levels of IL-2, IL-4, IL-5 and IFN-gamma were determined for CD3, CD44, CD49d, CD54, CD62L and CD102 lymphocytes from the bronchoalveolar lavage and spleen. To examine activation at the DNA level, lymphocytes expressing IL-2, IL-4, IL-5 or IFN-gamma were analysed for G2+M DNA content or phosphatidylserine expression (apoptosis). Trafficking of lymphocytes to the BAL was detected at day 5 p.i., peaked day 7 p.i., and predominately involved CD54(+)and CD102(+)lymphocytes expressing high levels of IL-2, IL-4, IL-5 and IFN-gamma. Lymphocytes expressing CD44(+), CD49d(+)and CD62L(lo)were also observed, however they expressed these cytokines to a lesser extent. DNA analysis of lymphocytes expressing IL-2 or IFN-gamma revealed higher G2'M levels compared to lymphocytes expressing IL-4 or IL-5, suggesting greater activation of Th(1)-type lymphocytes in the lung. These data demonstrate that RSV-induced pulmonary inflammation involves extensive cellular activation and cytokine expression, particularly by CD54(+)and CD102(+)lymphocytes in the lung.     

DNA immunization with Plasmodium falciparum serine repeat antigen: regulation of humoral immune response by coinoculation of cytokine expression plasmid

We immunized mice with plasmid expressing the 47-kDa amino-terminal domain of the Plasmodium falciparum serine repeat antigen (SERA) using gene gun and investigated humoral immune response to SERA antigen. Significant SERA-specific IgG was observed in BALB/c mice after immunization three times with SERA expression plasmid. Furthermore, these levels were increased by the coinoculation of cytokine (IFN-gamma, IL-4, GM-CSF, or IL-12) expression plasmid. In respect to the SERA-specific Ig subclasses, coinoculation of IFN-gamma, GM-CSF, or IL-12 expression plasmid increased the levels of SERA-specific IgG2a, and these were much higher than that in mice immunized with SERA expression plasmid alone. In contrast to the SERA-specific IgG2a, coinoculation of any cytokine expression plasmid did not change the levels of SERA-specific IgG1. These results indicate that cytokine expression plasmid enhances and regulates humoral immune response elicited by SERA DNA immunization.     

Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination.

Previously we showed that mice immunized with a vaccinia virus vector expressing the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene (vaccinia/gD) were protected against both lethal and latent infections with HSV-1 for at least 6 weeks after immunization (K. J. Cremer, M. Mackett, C. Wohlenberg, A. L. Notkins, and B. Moss, Science 228:737-740, 1985). In the experiments described here, we examined long-term immunity to HSV following vaccinia/gD vaccination, the effect of revaccination with vaccinia/gD, and the impact of previous immunity to vaccinia virus on immunization with the gD recombinant. Mice immunized with vaccinia/gD showed 100, 100, and 80% protection against lethal infection with HSV-1 at 18, 44, and 60 weeks postimmunization, respectively. Protection against latent trigeminal ganglionic infection was 70, 50, and 31% at 6, 41, and 60 weeks postvaccination, respectively. To study the effect of reimmunization on antibody levels, mice vaccinated with vaccinia/gD were given a second immunization (booster dose) 3 months after the first. These mice developed a 10-fold increase in neutralizing-antibody titer (221 to 2,934) and demonstrated a significant increase in protection against lethal HSV-1 challenge compared with animals that received only one dose of vaccinia/gD. To determine whether preexisting immunity to vaccinia virus inhibited the response to vaccination with vaccinia/gD virus, mice were immunized with a recombinant vaccinia virus vector expressing antigens from either influenza A or hepatitis B virus and were then immunized (2 to 3 months later) with vaccinia/gD. These mice showed reduced titers of neutralizing antibody to HSV-1 and decreased protection against both lethal and latent infections with HSV-1 compared with animals vaccinated only with vaccinia/gD. We conclude that vaccination with vaccinia/gD produces immunity against HSV-1 that lasts over 1 year and that this immunity can be increased by a booster but that prior immunization with a vaccinia recombinant virus expressing a non-HSV gene reduces the levels of neutralizing antibody and protective immunity against HSV-1 challenge.     

Protective and memory immunity to Histoplasma capsulatum in the absence of IL-10

We determined whether the absence of IL-10 in mice influenced protective and memory immunity to Histoplasma capsulatum. IL-10(-/-) mice cleared primary and secondary infection more rapidly than wild-type controls. Administration of mAb to TNF-alpha or IFN-gamma, but not GM-CSF, abrogated protection in naive IL-10(-/-) mice; mAb to TNF-alpha, but not IFN-gamma or GM-CSF, subverted protective immunity in secondary histoplasmosis. The inflammatory cell composition in IL-10(-/-) mice was altered in those given mAb to IFN-gamma or TNF-alpha. More Gr-1(+) and Mac-3(+) cells were present in lungs of IL-10(-/-) mice given mAb to IFN-gamma, and treatment with mAb to TNF-alpha sharply reduced the number of CD8(+) cells in lungs of IL-10(-/-) mice. We ascertained whether the lack of IL-10 modulated memory T cell generation or the protective function of cells. The percentage of CD3(+), CD44(high), CD62(low), and IFN-gamma(+) cells in IL-10(-/-) mice was higher than that of wild-type at day 7 but not day 21 or 49 after immunization. Fewer splenocytes from immunized IL-10(-/-) mice were required to mediate protection upon adoptive transfer into infected TCR alphabeta(-/-) mice. Hence, deficiency of IL-10 confers a salutary effect on the course of histoplasmosis, and the beneficial effects of IL-10 deficiency require endogenous TNF-alpha and/or IFN-gamma. Memory cell generation was transiently increased in IL-10(-/-) mice, but the protective function conferred by cells from these mice following immunization is strikingly more vigorous than that of wild-type.     

BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice

Cellular immune responses are required for protective immunity against Leishmania chagasi. Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens. Previous studies demonstrated that the L. chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L. chagasi infection. To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1). Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L. chagasi infection than LCR1 protein alone. BCG-LCR1 administered intraperitoneally did not protect. Splenocytes from mice immunized s.c. with either BCG-LCR1 or BCG-pMV261 and then infected with L. chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice. We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine.     

Vaccination with recombinant vaccinia viruses expressing ICP27 induces protective immunity against herpes simplex virus through CD4+ Th1+ T cells.

This study was designed to evaluate the efficacy and mechanisms of protection mediated by recombinant vaccinia viruses encoding immediate-early (IE) proteins of herpes simplex virus type 2 (HSV-2). Three mouse strains were immunized against the IE proteins ICP27, ICP0, and ICP4, and mice were challenged intracutaneously in the zosteriform model with HSV-2 strain MS. Protection was observed only following immunization with the ICP27 construct and then only in the BALB/c mouse strain. Protection in BALB/c mice was ablated by CD4+ T-cell suppression but remained intact in animals depleted of CD8+ T cells. Moreover, protection could be afforded to SCID nude recipients with CD4+ but not CD8+ T cells from ICP27-immunized mice. Only BALB/c mice developed a delayed-type hypersensitivity reaction to HSV-2, and in vitro measurements of humoral and cell-mediated immunity revealed response patterns to ICP27 and HSV that differed between protected BALB/c and unprotected mouse strains. Accordingly, BALB/c responses showed antigen-induced cytokine profiles dominated by type 1 cytokines, whereas C57BL/6 and C3H/HeN mice generated cytokine responses mainly of the type 2 variety. Our results may indicate that protection against zosterification is mainly mediated by CD4+ T cells that express a type 1 cytokine profile and that protective vaccines against HSV which effectively induce such T-cell responses should be chosen.     

Live attenuated influenza virus expressing human interleukin-2 reveals increased immunogenic potential in young and aged hosts

Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinant ca parent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8(+) T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8(+) T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8(+) T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups.     

Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice

We have generated recombinant influenza A viruses belonging to the H1N1 and H3N2 virus subtypes containing an insertion of the 137 C-terminal amino acid residues of the human immunodeficiency virus type 1 (HIV-1) Nef protein into the influenza A virus nonstructural-protein (NS1) reading frame. These viral vectors were found to be genetically stable and capable of growing efficiently in embryonated chicken eggs and tissue culture cells but did not replicate in the murine respiratory tract. Despite the hyperattenuated phenotype of influenza/NS-Nef viruses, a Nef and influenza virus (nucleoprotein)-specific CD8(+)-T-cell response was detected in spleens and the lymph nodes draining the respiratory tract after a single intranasal immunization of mice. Compared to the primary response, a marked enhancement of the CD8(+)-T-cell response was detected in the systemic and mucosal compartments, including mouse urogenital tracts, if mice were primed with the H1N1 subtype vector and subsequently boosted with the H3N2 subtype vector. In addition, Nef-specific serum IgG was detected in mice which were immunized twice with the recombinant H1N1 and then boosted with the recombinant H3N2 subtype virus. These findings may contribute to the development of alternative immunization strategies utilizing hyperattenuated live recombinant influenza virus vectors to prevent or control infectious diseases, e.g., HIV-1 infection.     

Immunogenicity and protection efficacy of replication-deficient influenza A viruses with altered NS1 genes

We explored the immunogenic properties of influenza A viruses with altered NS1 genes (NS1 mutant viruses). NS1 mutant viruses expressing NS1 proteins with an impaired RNA-binding function or insertion of a longer foreign sequence did not replicate in murine lungs but still were capable of inducing a Th1-type immune response resulting in significant titers of virus-specific serum and mucosal immunoglobulin G2 (IgG2) and IgA, but with lower titers of IgG1. In contrast, replicating viruses elicited high titers of serum and mucosal IgG1 but less serum IgA. Replication-deficient NS1 mutant viruses induced a rapid local release of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Moreover, these viruses also elicited markedly higher levels of IFN-alpha/beta in serum than the wild-type virus. Comparable numbers of virus-specific primary CD8(+) T cells were determined in all of the groups of immunized mice. The most rapid onset of the recall CD8(+)-T-cell response upon the wild-type virus challenge was detected in mice primed with NS1 mutant viruses eliciting high levels of cytokines. It is noteworthy that there was one NS1 mutant virus encoding NS1 protein with a deletion of 40 amino acids predominantly in the RNA-binding domain that induced the highest levels of IFN-alpha/beta, IL-6 and IL-1beta after infection. Mice that were immunized with this virus were completely protected from the challenge infection. These findings indicate that a targeted modification of the RNA-binding domain of the NS1 protein is a valuable technique to generate replication-deficient, but immunogenic influenza virus vaccines.     

Role of Interleukin-2 and Herpes Simplex Virus 1 in Central Nervous System Demyelination in Mice

We have reported previously that ocular infection of different strains of mice with recombinant herpes simplex virus 1 (HSV-1) constitutively expressing interleukin-2 (IL-2) provokes central nervous system (CNS) demyelination and optic neuropathy, as determined by changes in visual evoked cortical potentials and pathological changes in the optic nerve and CNS, whereas recombinant viruses expressing IL-4, gamma interferon, IL-12p35, IL-12p40, or IL-12p70 do not induce this neuropathy. The goal of this study was to dissect the mechanism underlying the interplay between the immune system (elevation of IL-2) and an environmental factor (infection with HSV-1) that elicits this pathology. Similar results were obtained upon delivery of IL-2 into the mouse brain using osmotic minipumps or injection of mice with recombinant IL-2 protein, IL-2 DNA, or IL-2 synthetic peptides prior to infection with wild-type (wt) HSV-1 strains McKrae and KOS. The critical role of IL-2 is further supported by our data, indicating that a single mutation at position T27A in IL-2 completely blocks the HSV-1-induced pathology. This study shows a novel model of autoimmunity in which viral infection and enhanced IL-2 cause CNS demyelination.