Simultaneous approach using systemic, mucosal and transcutaneous routes of immunization for development of protective HIV-1 vaccines.

Mucosal tissues are major sites of HIV entry and initial infection. Induction of a local mucosal cytotoxic T lymphocyte response is considered an important goal in developing an effective HIV vaccine. In addition, activation and recruitment of memory CD4(+) and CD8(+) T cells in systemic lymphoid circulation to mucosal effector sites might provide the firewall needed to prevent virus spread. Therefore a vaccine that generates CD4(+) and CD8(+) responses in both mucosal and systemic tissues might be required for protection against HIV. However, optimal routes and number of vaccinations required for the generation of long lasting CD4(+) and CD8(+) CTL effector and memory responses are not well understood especially for mucosal T cells. A number of studies looking at protective immune responses against diverse mucosal pathogens have shown that mucosal vaccination is necessary to induce a compartmentalized immune response including maximum levels of mucosal high-avidity CD8(+) CTL, antigen specific mucosal antibodies titers (especially sIgA), as well as induction of innate anti-viral factors in mucosa tissue. Immune responses are detectable at mucosal sites after systemic delivery of vaccine, and prime boost regimens can amplify the magnitude of immune responses in mucosal sites and in systemic lymphoid tissues. We believe that the most optimal mucosal and systemic HIV/SIV specific protective immune responses and innate factors might best be achieved by simultaneous mucosal and systemic prime and boost vaccinations. Similar principals of vaccination may be applied for vaccine development against cancer and highly invasive pathogens that lead to chronic infection.

Assessment of the relative therapeutic effects of vaccines on virus load and immune responses in small groups at several time points: efficacy of mucosal and subcutaneous polypeptide vaccines in rhesus macaques exposed to SHIV.

BACKGROUND: Due to the high cost, subject availability and ethical constraints, it is often critically important in pre-clinical and clinical studies to carry out an adequate statistical analysis of longitudinal multivariate data over several time points in trials in several small groups. OBJECTIVES: We aim to accurately assess and develop an appropriate distribution-free longitudinal model for an estimate of the comparative treatment effects of several biological factors in several small groups even if data sets should contain outlier measurements and censored values. This approach is used to evaluate the relative efficacy of mucosal and subcutaneous polypeptide vaccines in rhesus macaques exposed to SHIV. STUDY DESIGN: The algorithms of the non-parametric repeated measures ANOVA models [Mack GA. A quick and easy distribution-free test for main effects in a two-factor ANOVA. Communic Stat Part B: Simp Comp 1981;10:571-91; Akritas MG, Brunner, E. A unified approach to rank tests for mixed models. J Stat Plan Inference 1997;61:249-77; Brunner E, Puri ML. Nonparametric methods in factorial designs. Stat Pap 2001;42:1-52.] are described, programmed and assessed. The viral loads, CD4(+) and CD8(+) cell counts were analyzed at several time points in peripheral blood of the 11 MamuA(*)01 positive macaques intrarectally challenged with pathogenic SHIV-Ku2 and immunized intrarectally with synthetic HIV/SIV peptide vaccine. RESULTS: Using nonparametric ANOVA tests, we demonstrated with statistical significance that after intrarectal challenge with pathogenic SHIV-Ku2, intrarectally immunized monkeys expressed viral titers that fell below the level of detection in blood and intestine (which is a major reservoir of viral replication), whereas the subcutaneously immunized or control macaques had residual viraemia. Moreover, the proliferative response of T cells and both CD4(+) and CD8(+) cells were better preserved in intrarectally immunized animals. Robustness of the comparisons was confirmed by gradual removal of up to 50% of data points. CONCLUSION: Despite limited data, our analysis shows better preservation of both CD4(+) and CD8(+) cells in intrarectally immunized animals. The result is consistent with our hypothesis that mucosal immunization is more effective than systemic immunization and that an induction of specific CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates SHIV infection in non-human primates. Our analytical methodology can be applicable in comparative estimates of the different treatment-associated effects and their synergy for a variety of longitudinal data sets in small treatment groups.

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

High-affinity T helper epitope induces complementary helper and APC polarization, increased CTL, and protection against viral infection.

Natural viral proteins do not always make optimal vaccines. We have found that sequence modification to increase epitope affinity for class II MHC molecules (epitope enhancement) can improve immunogenicity. Here we show first that a higher-affinity helper epitope-enhanced HIV vaccine not only induces more cytotoxic T lymphocytes (CTLs), but also skews helper cells toward Th1 cytokine production and protects against HIV-1 recombinant vaccinia viral challenge. Furthermore, we elucidate a novel mechanism in which the higher-affinity vaccine induces dramatically more effective helper cells with a higher level of CD40L per helper cell and more positive cells, which in turn more effectively conditions dendritic cells (DCs) for CTL activation in a second culture. The improved helper cells also induce much greater IL-12 production by DCs, accounting for the reciprocal T helper polarization to Th1, and increase costimulatory molecule expression. Thus, increasing affinity for class II MHC results in a complementary interaction in which T helper and antigen-presenting cells polarize each other, as well as increase CTL, and provide greater vaccine efficacy against viral infection.

Activating CTL precursors to reveal CTL function without skewing the repertoire by in vitro expansion.

Detection of the functional CD8(+) CTL response usually requires in vitro restimulation. The differences between the CD8(+) CTL repertoire in freshly isolated precursor cells and CD8(+) CTL after short-term in vitro expansion have been generally assumed to be minimal, but have never been defined experimentally. Using staining with P18-I10/H-2D(d) tetramers and monoclonal antibodies (mAb) against Vbeta, we show the surprising result that there was significant skewing of the CD8(+) CTL repertoire after just 7 days of stimulation. In contrast, we found that overnight incubation of precursor cells with peptide allows the functional assessment of CD8(+) CTL (which cannot be detected ex vivo from freshly isolated cells) without changing the absolute number of antigen-specific CTL as measured by tetramer staining or the repertoire of TCR analyzed with mAb. This study affords a better understanding of the differences between the ex vivo and in vitro stimulated CTL repertoire, and provides an approach to reveal a more faithful representation of the functional in vivo CTL response without skewing of the repertoire of T cells detected.

Impairment of Gag-specific CD8(+) T-cell function in mucosal and systemic compartments of simian immunodeficiency virus mac251- and simian-human immunodeficiency virus KU2-infected macaques.

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.

Granulocyte-macrophage colony-stimulating factor expressed by recombinant respiratory syncytial virus attenuates viral replication and increases the level of pulmonary antigen-presenting cells.

An obstacle to developing a vaccine against human respiratory syncytial virus (RSV) is that natural infection typically does not confer solid immunity to reinfection. To investigate methods to augment the immune response, recombinant RSV (rRSV) was constructed that expresses murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) from a transcription cassette inserted into the G-F intergenic region. Replication of rRSV/mGM-CSF in the upper and lower respiratory tracts of BALB/c mice was reduced 23- to 74- and 5- to 588-fold, respectively, compared to that of the parental rRSV. Despite this strong attenuation of replication, the level of RSV-specific serum antibodies induced by rRSV/mGM-CSF was comparable to, or marginally higher than, that of the parental rRSV. The induction of RSV-specific CD8(+) cytotoxic T cells was moderately reduced during the initial infection, which might be a consequence of reduced antigen expression. Mice infected with rRSV/mGM-CSF had elevated levels of pulmonary mRNA for gamma interferon (IFN-gamma) and interleukin 12 (IL-12) p40 compared to animals infected by wild-type rRSV. Elevated synthesis of IFN-gamma could account for the restriction of RSV replication, as was observed previously with an IFN-gamma-expressing rRSV. The accumulation of total pulmonary mononuclear cells and total CD4(+) T lymphocytes was accelerated in animals infected with rRSV/mGM-CSF compared to that in animals infected with the control virus, and the level of IFN-gamma-positive or IL-4-positive pulmonary CD4(+) cells was elevated approximately twofold. The number of pulmonary lymphoid and myeloid dendritic cells and macrophages was increased up to fourfold in mice infected with rRSV/mGM-CSF compared to those infected with the parental rRSV, and the mean expression of major histocompatibility complex class II molecules, a marker of activation, was significantly increased in the two subsets of dendritic cells. Enhanced antigen presentation likely accounts for the maintenance of a strong antibody response despite reduced viral replication and would be a desirable property for a live attenuated rRSV vaccine.

Signals delivered through TCR instruct IL-12 receptor (IL-12R) expression: IL-12 and tumor necrosis factor-alpha synergize for IL-12R expression at low antigen dose.

Regulation of the IL-12 receptor (IL-12R) beta2 chain has been suggested to function as a molecular switch in determining T cell phenotype. However, because most studies have been carried out under conditions in which cell proliferation was occurring, it has been difficult to distinguish between instructive and selective mechanisms in regulating this key receptor. Here, in the course of trying to understand the mechanism for synergy between IL-12 and TNF-alpha in up-regulating IFN-gamma production, we find that when the stimulus through the TCR is too weak to induce cell proliferation, which would be needed for selection, IL-12 and TNF-alpha synergize to up-regulate not only IFN-gamma, but also the IL-12Rbeta2 chain, which triggers IFN-gamma production. Neither cytokine alone was sufficient. This observation held true both in the absence of antigen-presenting cells (APC), when the stimulus was anti-CD3 on plastic, and in the presence of APC presenting ovalbumin peptide to TCR-transgenic T cells. In contrast, when the TCR signal was stronger, no cytokines were necessary to up-regulate the IL-12R. Our results support the strength of signal model in instructing Th phenotype, and suggest both an instructive role and, later, through the production of IFN-gamma, a selective role, of this synergistic combination of cytokines in the preferential differentiation and expansion of Th1 cells.

Competitive inhibition in vivo and skewing of the T cell repertoire of antigen-specific CTL priming by an anti-peptide-MHC monoclonal antibody.

We have recently described a mAb, KP15, directed against the MHC-I/peptide molecular complex consisting of H-2D(d) and a decamer peptide corresponding to residues 311-320 of the HIV IIIB envelope glycoprotein gp160. When administered at the time of primary immunization with a vaccinia virus vector encoding gp160, the mAb blocks the subsequent appearance of CD8(+) CTL with specificity for the immunodominant Ag, P18-I10, presented by H-2D(d). This inhibition is specific for this particular peptide Ag; another H-2D(d)-restricted gp160 encoded epitope from a different HIV strain is not affected, and an H-2L(d)-restricted epitope encoded by the viral vector is also not affected. Using functional assays and specific immunofluorescent staining with multivalent, labeled H-2D(d)/P18-I10 complexes (tetramers), we have enumerated the effects of blocking of priming on the subsequent appearance, avidity, and TCR Vbeta usage of Ag-specific CTL. Ab blocking skews the proportion of high avidity cells emerging from immunization. Surprisingly, Vbeta7-bearing Ag-specific TCR are predominantly inhibited, while TCR of several other families studied are not affected. The ability of a specific MHC/peptide mAb to inhibit and divert the CD8(+) T cell response holds implications for vaccine design and approaches to modulate the immune response in autoimmunity.

Immunostimulatory DNA-based vaccines elicit multifaceted immune responses against HIV at systemic and mucosal sites.

Immunostimulatory DNA sequences (ISS, also known as CpG motifs) are pathogen-associated molecular patterns that are potent stimulators of innate immunity. We tested the ability of ISS to act as an immunostimulatory pathogen-associated molecular pattern in a model HIV vaccine using gp120 envelope protein as the Ag. Mice immunized with gp120 and ISS, or a gp120:ISS conjugate, developed gp120-specific immune responses which included: 1) Ab production; 2) a Th1-biased cytokine response; 3) the secretion of beta-chemokines, which are known to inhibit the use of the CCR5 coreceptor by HIV; 4) CTL activity; 5) mucosal immune responses; and 6) CD8 T cell responses that were independent of CD4 T cell help. Based on these results, ISS-based immunization holds promise for the development of an effective preventive and therapeutic HIV vaccine.

Mechanisms of cytokine synergy essential for vaccine protection against viral challenge.

The ability of cytokines to steer CD4(+) T(h) cell responses toward a T(h)1 or T(h)2 phenotype and enhance the magnitude of both CD8(+) cytotoxic T lymphocytes (CTL) and antibody responses has clearly been demonstrated by our lab and others, but the influence of cytokines on protective immune responses is much less clear. Here we show an essential role for CD4(+) T(h)1 helper cell induction and IFN-gamma production in protection from viral challenge with a recombinant vaccinia virus expressing HIV-1MN viral envelope glycoprotein gp160. Complete protection from viral challenge is achieved only when the triple combination of exogenous cytokines granulocyte macrophage colony stimulating factor (GM-CSF), IL-12 and tumor necrosis factor (TNF)-alpha are co-administered with the peptide vaccine. In vivo depletion of CD4(+) cells or immunization of IFN-gamma-deficient mice abrogates protection. GM-CSF, IL-12 and TNF-alpha also synergize for the enhanced induction of CTL; however, adoptive transfer of a CD8(+) CTL line afforded only partial protection in this viral challenge model. As a possible mechanism of in vivo protection we show that GM-CSF increases the percentage and activity of antigen-presenting dendritic cells in draining lymph nodes where the immune response is initiated. We further demonstrate synergy between IL-12 and the proinflammatory cytokine TNF-alpha in driving IFN-gamma production. Thus, a combination of IL-12 and TNF-alpha is essential for the optimal development of T(h)1 responses and help for CTL induction in BALB/c mice, and is complemented by a third cytokine, GM-CSF, which enhances antigen presentation.

Rabies virus-based vectors expressing human immunodeficiency virus type 1 (HIV-1) envelope protein induce a strong, cross-reactive cytotoxic T-lymphocyte response against envelope proteins from different HIV-1 isolates.

Novel viral vectors that are able to induce both strong and long-lasting immune responses may be required as effective vaccines for human immunodeficiency virus type 1 (HIV-1) infection. Our previous experiments with a replication-competent vaccine strain-based rabies virus (RV) expressing HIV-1 envelope protein from a laboratory-adapted HIV-1 strain (NL4-3) and a primary HIV-1 isolate (89.6) showed that RV-based vectors are excellent for B-cell priming. Here we report that cytotoxic T-lymphocyte (CTL) responses against HIV-1 gp160 are induced by recombinant RVs. Our results indicated that a single inoculation of mice with an RV expressing HIV-1 gp160 induced a solid and long-lasting memory CTL response specific for HIV-1 envelope protein. Moreover, CTLs from immunized mice were not restricted to the homologous HIV-1 envelope protein and were able to cross-kill target cells expressing HIV-1 gp160 from heterologous HIV-1 strains. These studies further suggest promise for RV-based vectors to elicit a persistent immune response against HIV-1 and their potential utility as efficacious anti-HIV-1 vaccines.

Antibodies directed against the MHC-I molecule H-2Dd complexed with an antigenic peptide: similarities to a T cell receptor with the same specificity.

alphabeta TCRs, which use an Ab-like structure to form a combining site, recognize molecular complexes consisting of peptides bound to MHC class I (MHC-I) or class II (MHC-II) molecules. To explore the similarities and differences between Ab and T cell recognition of similar structures, we have isolated two mAbs, KP14 and KP15, that specifically bind H-2D(d) complexed with an HIV envelope gp160-derived peptide, P18-I10. These Abs are MHC and peptide specific. Fine specificity of mAb binding was analyzed using a panel of synthetic peptides, revealing similarities between the mAb and a cloned TCR with the same specificity. These two mAbs used the same V(H) and J(H) gene segments, but different D, Vkappa, and Jkappa genes. Administered in vivo, mAb KP15 blocked the induction of CTL specific for recombinant vaccinia virus-encoded gp160, indicating its ability to bind endogenously generated MHC/peptide complexes. Analysis of the fine specificity of these mAbs in the context of their encoded amino acid sequences and the known three-dimensional structure of the H-2D(d)/P18-I10 complex suggests that they bind in an orientation similar to that of the TCR. Thus, the plasticity of the B cell receptor repertoire and the structural similarities among BCR and TCR allow Abs to effectively mimic alphabeta TCRs. Such mAbs may be useful in the therapeutic modulation of immune responses against infectious agents or harmful self Ags as well as in tracing steps in Ag processing.

Interplay of cytokines and adjuvants in the regulation of mucosal and systemic HIV-specific CTL.

We examined the interplay between cytokines and adjuvants to optimize the induction of CTL by a mucosal HIV peptide vaccine. We show synergy between IL-12 and GM-CSF when administered together with the HIV peptide PCLUS3-18IIIB and cholera toxin (CT) in the induction of CTL activity and protection against mucosal viral transmission. Further, we examine the efficacy of mutant Escherichia coli labile toxin, LT(R192G), as a less toxic adjuvant than CT. LT(R192G) was as effective as or more effective than CT at inducing a mucosal CTL response. Moreover, LT(R192G) was as effective without IL-12 as CT was when combined with IL-12, and the response elicited by LT(R192G) with the vaccine was not further enhanced by the addition of IL-12. GM-CSF synergized with LT(R192G) without exogenous IL-12. Therefore, LT(R192G) may induce a more favorable cytokine response by not inhibiting IL-12 production. In particular, less IL-4 is made after LT(R192G) than CT immunization, and the response is less susceptible to anti-IL-12 inhibition. Thus, the choice of mucosal adjuvant affects the cytokine environment, and the mucosal response and protection can be enhanced by manipulating the cytokine environment with synergistic cytokine combinations incorporated in the vaccine.

Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections.

We used several approaches to develop enhanced vaccines for chronic viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). 1) Selected epitopes were used to avoid potentially harmful immune responses. 2) Linkage between helper and cytotoxic T-lymphocyte (CTL) epitopes was found to be important. 3) We developed an "epitope enhancement" approach modifying the sequences of epitopes to make more potent vaccines, including examples for HIV and HCV epitopes presented by murine class II and human class I major histocompatibility complex (MHC) molecules. 4) CTL avidity was found to be important for clearing viral infections in vivo, and the mechanism was examined. High-avidity CTLs, however, were found to undergo apoptosis when confronted with high-density antigen, through a mechanism involving tumor necrosis factor (TNF), TNF-RII, and a permissive state induced through the T-cell receptor. 5) We employed cytokines in the adjuvant to steer immune responses toward desired phenotypes, and showed synergy between cytokines. 6) Intrarectal immunization with peptide vaccine induced mucosal and systemic CTL. Local mucosal CTL were found to be critical for resistance to mucosal viral transmission and this resistance was enhanced with mucosally delivered interleukin-12. 7) We used an asymmetry in induction of mucosal and systemic immune responses to circumvent pre-existing vaccinia immunity for use of recombinant vaccinia vaccines.

NK and CTL recognition of a single chain H-2Dd molecule: distinct sites of H-2Dd interact with NK and TCR.

We generated transgenic mice expressing a single-chain beta2-microglobulin (beta2m)-H-2Dd. The cell-surface beta2m-H-2Dd molecule was expressed on a beta2m-deficient background and reacted with appropriate mAbs. It was of the expected m.w. and directed the normal development of CD8+ T cells in the thymus of a broad TCR repertoire. It also presented both exogenously provided and endogenous peptide Ags to effector CD8+ T cells. In tests of NK cell education and function, it failed to reveal any interaction with NK cells, suggesting that the site of the interaction of NK receptors with H-2Dd was disrupted. Thus, the sites of TCR and NK receptor interaction with H-2Dd are distinct, an observation consistent with independent modes of TCR and NK receptor evolution and function.

Mucosal vaccination overcomes the barrier to recombinant vaccinia immunization caused by preexisting poxvirus immunity.

Overcoming preexisting immunity to vaccinia virus in the adult population is a key requirement for development of otherwise potent recombinant vaccinia vaccines. Based on our observation that s.c. immunization with vaccinia induces cellular and antibody immunity to vaccinia only in systemic lymphoid tissue and not in mucosal sites, we hypothesized that the mucosal immune system remains naive to vaccinia and therefore amenable to immunization with recombinant vaccinia vectors despite earlier vaccinia exposure. We show that mucosal immunization of vaccinia-immune BALB/c mice with recombinant vaccinia expressing HIV gp160 induced specific serum antibody and strong HIV-specific cytotoxic T lymphocyte responses. These responses occurred not only in mucosal but also in systemic lymphoid tissue, whereas systemic immunization was ineffective under these circumstances. In this context, intrarectal immunization was more effective than intranasal immunization. Boosting with a second dose of recombinant vaccinia was also more effective via the mucosal route. The systemic HIV-specific cytotoxic T lymphocyte response was enhanced by coadministration of IL-12 at the mucosal site. These results also demonstrate the independent compartmentalization of the mucosal versus systemic immune systems and the asymmetric trafficking of lymphocytes between them. This approach to circumvent previous vaccinia immunity may be useful for induction of protective immunity against infectious diseases and cancer in the sizable populations with preexisting immunity to vaccinia from smallpox vaccination.

Induction of a mucosal cytotoxic T-lymphocyte response by intrarectal immunization with a replication-deficient recombinant vaccinia virus expressing human immunodeficiency virus 89.6 envelope protein.

To improve the safety of recombinant vaccinia virus vaccines, modified vaccinia virus Ankara (MVA) has been employed, because it has a replication defect in most mammalian cells. Here we apply MVA to human immunodeficiency virus type 1 (HIV-1) vaccine development by incorporating the envelope protein gp160 of HIV-1 primary isolate strain 89.6 (MVA 89.6) and use it to induce mucosal cytotoxic-T-lymphocyte (CTL) immunity. In initial studies to define a dominant CTL epitope for HIV-1 89.6 gp160, we mapped the epitope to a sequence, IGPGRAFYAR (from the V3 loop), homologous to that recognized by HIV MN loop-specific CTL and showed that HIV-1 MN-specific CTLs cross-reactively recognize the corresponding epitope from strain 89.6 presented by H-2Dd. Having defined the CTL specificity, we immunized BALB/c mice intrarectally with recombinant MVA 89.6. A single mucosal immunization with MVA 89.6 was able to elicit long-lasting antigen-specific mucosal (Peyer's patch and lamina propria) and systemic (spleen) CTL responses as effective as or more effective than those of a replication-competent vaccinia virus expressing 89.6 gp160. Immunization with MVA 89.6 led to (i) the loading of antigen-presenting cells in vivo, as measured by the ex vivo active presentation of the P18-89.6 peptide to an antigen-specific CTL line, and (ii) the significant production of the proinflammatory cytokines (interleukin-6 and tumor necrosis factor alpha) in the mucosal sites. These results indicate that nonreplicating recombinant MVA may be at least as effective for mucosal immunization as replicating recombinant vaccinia virus.

Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge.

Mucosal tissues are major sites of HIV entry and initial infection. Thus, the induction of a mucosal cytotoxic T lymphocyte (CTL) response is an important feature for an effective HIV vaccine. However, little is known about approaches to induce such a protective CTL response in the mucosa. Here for the first time we show that intrarectal immunization with a synthetic, multideterminant HIV peptide plus cholera toxin adjuvant induced long-lasting, antigen-specific CTL memory in both the inductive (Peyer's patch) and effector (lamina propria) mucosal sites, as well as in systemic sites (spleen), whereas systemic immunization induced specific CTL only in the spleen. Cholera toxin adjuvant, while enhancing the response, was not essential. The CTL recognized target cells either pulsed with HIV peptide or expressing endogenous whole envelope glycoprotein of Mr 160,000 (gp160). Exploring the requirements for CTL induction, we show that mucosal CTL responses are both interleukin 12 and interferon-gamma dependent by using antibody-treated and knock-out mice. Finally, to determine whether a mucosal response is actually protective against local mucosal challenge with virus, we show that intrarectal immunization with the synthetic HIV peptide vaccine protected mice against infection via mucosal challenge with a recombinant vaccinia virus expressing HIV-1IIIB gp160. These studies provide an approach to development of an HIV vaccine that induces CTL immunity in the mucosal and systemic immune systems and protects against mucosal infection with a virus expressing HIV-1 gp160.

Late T cell deficiency in victims of the Chernobyl radiation accident: possible mechanisms of induction.

T cell number, serum concentrations of thymic hormones and anti-epithelial autoantibodies were studied in people affected at Chernobyl NPP. Group 1 took part in the clearing-up operation and had no clinical manifestations of acute radiation sickness. Group 2 worked at the NPP during the accident; they survived acute radiation sickness (degree I-II, subgroup 2a; degree III-IV, subgroup 2b). The total doses of external radiation were 0.1-0.5 Gy in group 1, up to 4 Gy in subgroup 2a and up to 9 Gy in subgroup 2b. Total T cell number, serum thymic activity and alpha 1-thymosin concentration were decreased in all groups of affected persons. CD8+ cell number decreased only in group 1; CD4+ cell number in subgroup 2b. A decrease in thymic hormone level was most prominent in subgroup 2b. The titres of anti-epithelial antibodies were increased in all groups of affected persons independently of radiation dose. The titres were higher in patients with subnormal levels of alpha 1-thymosin. It has been proposed that radiation alters the function of thymic epithelial cells by direct action and/or through indirect mechanisms including participation of autoantibodies. The observed complex of alterations is similar to that in the normal process of immunological ageing.