A nonstructural viral protein expressed by a recombinant vaccinia virus protects against lethal cytomegalovirus infection.

The nonstructural immediate-early protein pp89 of murine cytomegalovirus (MCMV) is the first viral protein synthesized after infection and has a regulatory function in viral gene expression. Despite its localization in the nucleus of infected cells, pp89 is also the dominant antigen recognized by MCMV-specific cytolytic T lymphocytes. The recombinant vaccinia virus MCMV-ieI-VAC, which expresses pp89, was used to study the capacity of this protein to induce protective immunity in BALB/c mice. Vaccination with MCMV-ieI-VAC induced a long-lasting immunity that protected mice against challenge with a lethal dose of MCMV but did not prevent infection and morbidity. In vivo depletion of CD8+ T lymphocytes before challenge completely abrogated the protective immunity. CD8+ T lymphocytes derived from MCMV-ieI-VAC-primed donors and adoptively transferred into sublethally irradiated and MCMV-infected recipients were found to limit viral replication in host tissues, whereas CD4+ T lymphocytes and pp89-specific antiserum had no protective effect. The data demonstrate for the first time that a single nonstructural viral protein can confer protection against a lethal cytolytic infection and that this immunity is entirely mediated by the CD8+ subpopulation of T lymphocytes.     

Immunoglobulin G, Plasma Cells, and Lymphocytes in the Murine Vagina after Vaginal or Parenteral Immunization with Attenuated Herpes Simplex Virus Type 2

This investigation evaluated immunity to vaginal herpes simplex virus type 2 (HSV-2) infection after local or parenteral immunization with attenuated HSV-2. Vaginal immunization induced sterilizing immunity against challenge with a high dose of wild-type virus, whereas parenteral immunizations protected against neurologic disease but did not entirely prevent infection of the vagina. Vaginal immunization caused 86- and 31-fold increases in the numbers of immunoglobulin G (IgG) plasma cells in the vagina at 6 weeks and 10 months after immunization, whereas parenteral immunizations did not increase plasma cell numbers in the vagina. Vaginal secretion/serum titer ratios and specific antibody activities in vaginal secretions and serum indicated that IgG viral antibody was produced in the vagina and released into vaginal secretions at 6 weeks and 10 months after vaginal immunization but not after parenteral immunizations. In contrast to the case for plasma cells, the numbers of T and B lymphocytes in the vagina were similar in vaginally and parenterally immunized mice. Also, lymphocyte numbers in the vagina were markedly but similarly increased by vaginal challenge with HSV-2 in both vaginally and parenterally immunized mice. Lymphocyte recruitment to the vagina after virus challenge appeared to involve memory lymphocytes, because it was not observed in nonimmunized mice. Thus, local vaginal immunization with attenuated HSV-2 increased the number of IgG plasma cells in the vagina and increased vaginal secretion/serum titer ratios to 3.0- to 4.7-fold higher than in parenterally immunized groups but caused little if any selective homing of T and B lymphocytes to the vagina.     

Global dysfunction of CD4 T-lymphocyte cytokine expression in simian-human immunodeficiency virus/SIV-infected monkeys is prevented by vaccination

Human immunodeficiency virus type 1 infection results in a dysfunction of CD4(+) T lymphocytes. The intracellular events contributing to that CD4(+) T-lymphocyte dysfunction remain incompletely elucidated, and it is unclear whether aspects of that dysfunction can be prevented. The present studies were pursued in a rhesus monkey model of AIDS to explore these issues. Loss of the capacity of peripheral blood CD4(+) T lymphocytes to express cytokines was first detected in simian immunodeficiency virus-infected monkeys during the peak of viral replication during primary infection and persisted thereafter. Moreover, infected monkeys with progressive disease had peripheral blood CD4(+) T lymphocytes that expressed significantly less cytokine than infected monkeys that had undetectable viral loads and intact CD4(+) T-lymphocyte counts. Importantly, CD4(+) T lymphocytes from vaccinated monkeys that effectively controlled the replication of a highly pathogenic immunodeficiency virus isolate following a challenge had a preserved functional capacity. These observations suggest that an intact cytokine expression capacity of CD4(+) T lymphocytes is associated with stable clinical status and that effective vaccines can mitigate against CD4(+) T-lymphocyte dysfunction following an AIDS virus infection.     

Virus-specific memory and effector T lymphocytes exhibit different cytokine responses to antigens during experimental murine respiratory syncytial virus infection.

Mice sensitized to the G (attachment) or F (fusion) glycoproteins of respiratory syncytial virus (RSV) expressed different patterns of cytokine production and lung pathology when challenged by intranasal infection with RSV. Five days after challenge, mice sensitized to G glycoprotein produced high levels of interleukin-4 (IL-4) and IL-5 in the lungs and spleens and developed extensive pulmonary eosinophilia, while mice sensitized to F glycoprotein produced IL-2 and developed a mononuclear cell infiltration. Memory lymphocytes isolated 2 weeks after intranasal challenge of mice primed to the G or F glycoprotein secreted only IL-2 and gamma interferon (IFN-gamma) when stimulated with RSV. IL-4 and IL-5 production characteristic of Th2-type effectors in the lung was observed only after multiple rounds of in vitro stimulation of RSV G-specific memory T lymphocytes with antigen. Also IFN-gamma production appeared to play only a minor role in the expression of pulmonary pathology characteristic of Th1 or Th2 T-lymphocyte responses, because mice genetically deficient in IFN-gamma production by gene disruption displayed the same pattern of pulmonary inflammation to RSV infection after priming to RSV F or G as conventional mice. These results suggest that effector T lymphocytes exhibit a different pattern of cytokine production than memory T-lymphocyte precursors precommitted to a Th1 or Th2 pattern of differentiation. Furthermore, these observations raise the possibility that the cytokine response of human memory T lymphocytes after a single exposure to antigen in vitro may not accurately reflect the cytokine response of differentiated effector T lymphocytes at the site of infection in vivo.     

异型流感病毒感染相关T淋巴细胞增殖活性及IFN-γ阳性Ｔ淋巴细胞水平研究

为探讨机体异型流感病毒间交叉保护作用机制,将实验动物随机分成实验组和对照组,测定异型流感病毒感染后病毒载量,T淋巴细胞增殖活性和IFN-γ阳性CD3＋CD8＋及CD3＋CD4＋淋巴细胞水平的变化。结果显示,异型流感病毒感染后产生的交叉免疫应答反应可能与T淋巴细胞增殖有关;与CTL及Th1类淋巴细胞水平相关,并有时间限制性;IL-2可以加强异型流感病毒感染后IFN-γ阳性CD3＋CD8＋淋巴细胞水平。本研究为制备能够抵御变异流感病毒感染的疫苗提供了理论基础。     

Vaginal protection and immunity after oral immunization of mice with a novel vaccine strain of Listeria monocytogenes expressing human immunodeficiency virus type 1 gag

Natural transmission of human immunodeficiency virus (HIV) occurs at mucosal surfaces. During acute infection, intestinal and other mucosae are preferential sites of virus replication and rapidly become depleted of CD4(+) T cells. Therefore, mucosal immunity may be critical to control both initial infection and the massive early spread of virus. An attenuated D-alanine-requiring strain of the oral intracellular microorganism Listeria monocytogenes expressing HIV type 1 gag was shown to induce protective cell-mediated immunity in mice against viruses that express HIV gag when immunization occurs in the presence of a transient supply of D-alanine. In this study, we examined the efficacy of new attenuated strains that are able to synthesize d-alanine from a heterologous dal gene tightly regulated by an actA-promoted resolvase recombination system. In the absence of d-alanine, Gag-specific cytotoxic T lymphocytes (CTLs) were induced systemically after intravenous immunization, and one strain, Lmdd-gag/pARS, induced strong dose-dependent Gag-specific CTLs after oral immunization. A significant level of Gag-specific CD8(+) T cells was induced in the mucosal-associated lymphoid tissues (MALTs). Upon intravaginal challenge of these orally immunized mice with recombinant vaccinia virus (rVV) expressing HIV gag, gamma interferon- and tumor necrosis factor alpha-secreting Gag-specific CD8(+) T cells were dramatically increased in the spleen and MALTs. Oral immunization with Lmdd-gag/pARS led to complete protection against vaginal challenge by a homologous clade B gag-expressing rVV. In addition, strong cross-clade protection was seen against clades A and C and partial protection against clade G gag-expressing rVV. These results suggest that Lmdd-gag/pARS may be considered as a novel vaccine candidate for use against HIV/AIDS.     

Sustained viremia suppression by SHIVSF162P3CN-recalled effector-memory CD8+ T cells after PD1-based vaccination

HIV-1 functional cure requires sustained viral suppression without antiretroviral therapy. While effector-memory CD8⁺ T lymphocytes are essential for viremia control, few vaccines elicit such cellular immunity that could be potently recalled upon viral infection. Here, we investigated a program death-1 (PD1)-based vaccine by fusion of simian immunodeficiency virus capsid antigen to soluble PD1. Homologous vaccinations suppressed setpoint viremia to undetectable levels in vaccinated macaques following a high-dose intravenous challenge by the pathogenic SHIV_SF162P3CN. Poly-functional effector-memory CD8⁺ T cells were not only induced after vaccination, but were also recalled upon viral challenge for viremia control as determined by CD8 depletion. Vaccine-induced effector memory CD8⁺ subsets displayed high cytotoxicity-related genes by single-cell analysis. Vaccinees with sustained viremia suppression for over two years responded to boost vaccination without viral rebound. These results demonstrated that PD1-based vaccine-induced effector-memory CD8⁺ T cells were recalled by AIDS virus infection, providing a potential immunotherapy for functional cure.     

Do cytotoxic T lymphocytes clear some HIV/SIV infections?

Early work on the roles of cytotoxic T lymphocytes (CTLs) in acute viral infections in animal models showed that i) the clearance of virus coincided with the increase in CTL activity rather than specific antibody levels, ii) transfer of CTLs after infection could protect from a lethal dose of virus, and iii) in primed, compared to naive, animals, CTL activity appeared 1–3 days earlier after a challenge infection. There is now a series of findings with individuals who have been exposed to HIV but are HIV-seronegative that suggest a protective role for CTLs. Usually after in vitro culture, HIV-specific CTLs have been isolated from i) infants born of infected mothers, ii) long-time partners of HIV-infected people, iii) some prostitutes in Africa, and iv). Most recently, 7/20 seronegative health care workers exposed once to HIV have been shown to possess HIV env-specific CTLs. The findings suggest that CTLs (a type I T cell response) may rapidly clear a low dose of HIV. Experiments with SIV are proposed that may provide more direct supporting data for this possibility.     

Gag-Specific Cellular Immunity Determines In Vitro Viral Inhibition and In Vivo Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8(+) T lymphocytes from vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge. We observed that CD8(+) lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4(+) and CD8(+) T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.     

Replication of dengue-2 virus in cultured human lymphoblastoid cells and subpopulations of human peripheral leukocytes.

We studied the susceptibility of four human lymphoblastoid cell lines (HCL) and of subpopulations of circulating peripheral human leukocytes to dengue-2 virus infection. HCL with B cell characteristics (Raji, Wil 2WT, 8866), B-type peripheral lymphocytes, and macrophages were productively infected by dengue-2 virus. In contrast, an HCL with T cell characteristics (MOLT-4), T type peripheral lymphocytes, and polymorphonuclear (PMN) cells did not become infected and replicate dengue-2 virus. PMN cells did not adsorb dengue-2 virus, suggesting lack of viral receptors. However, T-type cultured lymphoblasts and T-type peripheral lymphocytes adsorbed dengue-2 virus, suggesting that the block in viral replication involves some stage of infection occurring after adsorption. Permissiveness of B-type HCL to dengue-2 virus infection was dependent on the virus seed used but the virus titers obtained among the susceptible HCL varied. HCL infected persistently with dengue-2 virus have been established. Human peripheral lymphocytes inoculated after cultivation for 3 days in complete medium alone or complete medium supplemented with mitogens replicated dengue-2 virus. In contrast, unstimulated peripheral lymphocytes inoculated immediately after isolation adsorbed dengue-2 but did not support its replication. Mitogen-treated and untreated macrophages replicated dengue-2 virus equally well. The efficiency of dengue-2 virus replication by macrophages was higher than that of peripheral lymphocytes but lower than that of HCL.     

Gamma delta T cells facilitate adaptive immunity against West Nile virus infection in mice

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and gammadelta T cells are involved in the protective immune response against viral challenge. We have now examined whether gammadelta T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRdelta(-/-) mice survived primary infection with WN virus compared with 80-85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of gammadelta T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, gammadelta T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8(+) T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8(+) T cells from TCRdelta(-/-) mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that gammadelta T cells directly link innate and adaptive immunity during WN virus infection.     

Virus-specific cellular immune correlates of survival in vaccinated monkeys after simian immunodeficiency virus challenge

Understanding the characteristics of the virus-specific T-lymphocyte response that will confer optimal protection against the clinical progression of AIDS will inform the development of an effective cellular immunity-based human immunodeficiency virus vaccine. We have recently shown that survival in plasmid DNA-primed/recombinant adenovirus-boosted rhesus monkeys that are challenged with the simian immunodeficiency virus SIVmac251 is associated with the preservation postchallenge of central memory CD4(+) T lymphocytes and robust gamma interferon (IFN-gamma)-producing SIV-specific CD8(+) and CD4(+) T-lymphocyte responses. The present studies were initiated to extend these observations to determine which virus-specific T-lymphocyte subpopulations play a primary role in controlling disease progression and to characterize the functional repertoire of these cells. We show that the preservation of the SIV-specific central memory CD8(+) T-lymphocyte population and a linked SIV-specific CD4(+) T-lymphocyte response are associated with prolonged survival in vaccinated monkeys following challenge. Furthermore, we demonstrate that SIV-specific IFN-gamma-, tumor necrosis factor alpha-, and interleukin-2-producing T lymphocytes are all comparably associated with protection against disease progression. These findings underscore the contribution of virus-specific central memory T lymphocytes to controlling clinical progression in vaccinated individuals following a primate immunodeficiency virus infection.     

Effect of murine cytomegalovirus on the in vitro responses of T and B cells to mitogens.

Both in vitro and in vivo murine cytomegalovirus (MCMV) infection depressed the responses of lymphocytes to both B and T cell mitogens. The possibilities that macrophages or nonspecific T cell inhibition of B cells might account for the depressed responses were eliminated. In vitro data suggested that B cell responses are more susceptible to this depression than T cell responses. The possibility that the depression of T cell responses is not a direct effect of viral infection of lymphocytes is discussed. To investigate further the interaction between B and T lymphocytes and MCMV, mice with B and T cell deficiences were studied. A comparison of the susceptibility of athymic Nu/Nu mice and T cell competent Nu/+ littermates to MCMV showed that the LD50 for Nu/Nu mice is 10-fold lower than that for Nu/+ mice, but Nu/+ mice given an LD50 of virus died much sooner after infection than Nu/Nu mice given an LD50. Pathogenic mechanisms responsible for death may be different in these two groups of mice. Similarly the MCMV LD50 for B cell-deficient mice (treated with goat anti-mouse IgM serum) was 10-fold lower than the LD50 for mice treated with normal goat serum, but given an LD50 of virus, the latter died sooner after infection than the former. In contrast, there was little difference between the LD50 or time of death after MCMV infection of CBA x DBA F1 male mice (which are deficient in their response to thymic independent antigens) and their normal littermates, the CBA x DBA F1 female mice.     

Priming with cold-adapted influenza A does not prevent infection but elicits long-lived protection against supralethal challenge with heterosubtypic virus

We show in this study several novel features of T cell-based heterosubtypic immunity against the influenza A virus in mice. First, T cell-mediated heterosubtypic protection against lethal challenge can be generated by a very low priming dose. Second, it becomes effective within 5-6 days. Third, it provides protection against a very high dose challenge for >70 days. Also novel is the finding that strong, long-lasting, heterosubtypic protection can be elicited by priming with attenuated cold-adapted strains. We demonstrate that priming does not prevent infection of the lungs following challenge, but leads to earlier clearance of the virus and 100% survival after otherwise lethal challenge. Protection is dependent on CD8 T cells, and we show that CD4 and CD8 T cells reactive to conserved epitopes of the core proteins of the challenge virus are present after priming. Our results suggest that intranasal vaccination with cold-adapted, attenuated live virus has the potential to provide effective emergency protection against emerging influenza strains for several months.     

Rabies virus neuritic paralysis: immunopathogenesis of nonfatal paralytic rabies.

Two pathogenetically distinct disease manifestations are distinguished in a murine model of primary rabies virus infection with the Evelyn-Rokitnicky-Abelseth strain, rabies virus neuritic paralysis (RVNP) and fatal encephalopathogenic rabies. RVNP develops with high incidence in immunocompetent mice after intraplantar infection as a flaccid paralysis restricted to the infected limb. The histopathologic correlate of this monoplegia is a degeneration of the myelinated motor neurons of the peripheral nerve involved. While, in this model, fatal encephalopathogenic rabies develops only after depletion of the CD4 subset of T lymphocytes and without contribution of the CD8 subset, RVNP is identified as an immunopathological process in which both the CD4 and CD8 subsets of T lymphocytes are critically implicated.     

Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response.

After intravenous infection of mice, lymphocytic choriomeningitis virus multiplied in spleens and livers, attaining highest concentrations on days 4 to 6. The subsequent clearance was as rapid, and 8 to 10 days after inoculation, infectivity was usually below detectability. During the effector phase of virus elimination, both cytotoxic T-cell (CTL) activity and the number of cells producing antiviral antibodies were high. Monoclonal antibodies directed against T lymphocytes and T-lymphocyte subsets were inoculated once intravenously 5, 6, or 7 days after infection of the animals, and the effects on antiviral immune responses, as well as on elimination of virus from the organs, were determined. Treatment with anti-Thy-1 and anti-Lyt-2 antibodies blocked elimination of the virus and profoundly diminished the activity of spleen CTLs but reduced the antibody response partially (anti-Thy-1) or increased it (anti-Lyt-2). In contrast, treatment with the anti-L3T4 antibody had essentially no effect on either virus elimination or CTL response but abolished antibody production. We conclude that Lyt-2+ (cytotoxic-suppressive) T lymphocytes are needed for elimination of the virus and also regulate the humoral response but that antiviral antibodies are not essential for control of the infection.     

Role of T cells in virus control and disease after infection with pneumonia virus of mice

Infection of mice with pneumonia virus of mice (PVM) is used as a natural host experimental model for studying the pathogenesis of infection with the closely related human respiratory syncytial virus. We analyzed the contribution of T cells to virus control and pathology after PVM infection. Control of a sublethal infection with PVM strain 15 in C57BL/6 mice was accompanied by a 100-fold increase in pulmonary cytotoxic T lymphocytes, 20% of which were specific for PVM. T-cell-deficient mice failed to eliminate PVM and became virus carriers in the absence of the clinical or histopathological signs of pneumonia that occurred after infection of control mice. Mice with limited T-cell numbers did not achieve virus control without weight loss, indicating that T-cell-mediated virus control was closely linked to immunopathology. Both CD4 and CD8 T cells independently contributed to virus elimination and disease. Virus control and disease were similar in the absence of perforin, gamma interferon, or tumor necrosis factor alpha. Interestingly, disease and mortality after lethal high-dose PVM infection were independent of T cells. These data illustrate a key role for T cells in control of PVM infection and demonstrate that both T-cell-dependent and -independent pathways contribute to disease in a viral dose-dependent fashion.     

Rabies virus infects mouse and human lymphocytes and induces apoptosis.

Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response.     

Simian-human immunodeficiency virus SHIV89.6-induced protection against intravaginal challenge with pathogenic SIVmac239 is independent of the route of immunization and is associated with a combination of cytotoxic T-lymphocyte and alpha interferon responses

Attenuated primate lentivirus vaccines provide the most consistent protection against challenge with pathogenic simian immunodeficiency virus (SIV). Thus, they provide an excellent model to examine the influence of the route of immunization on challenge outcome and to study vaccine-induced protective anti-SIV immune responses. In the present study, rhesus macaques were immunized with live nonpathogenic simian-human immunodeficiency virus (SHIV) 89.6 either intravenously or mucosally (intranasally or intravaginally) and then challenged intravaginally with pathogenic SIVmac239. The route of immunization did not affect mucosal challenge outcome after a prolonged period of systemic infection with the nonpathogenic vaccine virus. Further, protection from the SIV challenge was associated with the induction of multiple host immune effector mechanisms. A comparison of immune responses in vaccinated-protected and vaccinated-unprotected animals revealed that vaccinated-protected animals had higher frequencies of SIV Gag-specific cytotoxic T lymphocytes and gamma interferon (IFN-gamma)-secreting cells during the acute phase postchallenge. Vaccinated-protected animals also had a more pronounced increase in peripheral blood mononuclear cell IFN-alpha mRNA levels than did the vaccinated-unprotected animals in the first few weeks after challenge. Thus, innate as well as cellular anti-SIV immune responses appeared to contribute to the SHIV89.6-induced protection against intravaginal challenge with pathogenic SIVmac239.     

The contribution of systemic and pulmonary immune effectors to vaccine-induced protection from H5N1 influenza virus infection

Live attenuated influenza vaccines (LAIVs) are effective in providing protection against influenza challenge in animal models and in preventing disease in humans. We previously showed that LAIVs elicit a range of immune effectors and that successful induction of pulmonary cellular and humoral immunity in mice requires pulmonary replication of the vaccine virus. An upper respiratory tract immunization (URTI) model was developed in mice to mimic the human situation, in which the vaccine virus does not replicate in the lower respiratory tract, allowing us to assess the protective efficacy of an H5N1 LAIV against highly pathogenic H5N1 virus challenge in the absence of significant pulmonary immunity. Our results show that, after one dose of an H5N1 LAIV, pulmonary influenza-specific lymphocytes are the main contributors to clearance of challenge virus from the lungs and that contributions of influenza-specific enzyme-linked immunosorbent assay (ELISA) antibodies in serum and splenic CD8(+) T cells were negligible. Complete protection from H5N1 challenge was achieved after two doses of H5N1 LAIV and was associated with maturation of the antibody response. Although passive transfer of sera from mice that received two doses of vaccine prevented lethality in naive recipients following challenge, the mice showed significant weight loss, with high pulmonary titers of the H5N1 virus. These data highlight the importance of mucosal immunity in mediating optimal protection against H5N1 infection. Understanding the requirements for effective induction and establishment of these protective immune effectors in the respiratory tract paves the way for a more rational and effective vaccine approach in the future.