Myd88 Is Required for an Antibody Response to Retroviral Infection

Although retroviruses have been extensively studied for many years, basic questions about how retroviral infections are detected by the immune system and which innate pathways are required for the generation of immune responses remain unanswered. Defining these pathways and how they contribute to the anti-retroviral immune responses would assist in the development of more effective vaccines for retroviral pathogens such as HIV. We have investigated the roles played by CD11c⁺ dendritic cells (DCs) and by Toll-like receptor (TLR) signaling pathways in the generation of an anti-retroviral immune response against a mouse retroviral pathogen, Friend murine leukemia virus (F-MLV). Specific deletion of DCs during F-MLV infection caused a significant increase in viral titers at 14 days post-infection, indicating the importance of DCs in immune control of the infection. Similarly, Myd88 knockout mice failed to control F-MLV, and sustained high viral titers (10⁷ foci/spleen) for several months after infection. Strikingly, both DC-depleted mice and Myd88 knockout mice exhibited only a partial reduction of CD8⁺ T cell responses, while the IgG antibody response to F-MLV was completely lost. Furthermore, passive transfer of immune serum from wild-type mice to Myd88 knockout mice rescued control of F-MLV. These results identify TLR signaling and CD11c⁺ DCs as playing critical roles in the humoral response to retroviruses.     

Immunogenic Profiling in Mice of a HIV/AIDS Vaccine Candidate (MVA-B) Expressing Four HIV-1 Antigens and Potentiation by Specific Gene Deletions

Background

The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function.

Methodology/Principal Findings

In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1β, respectively (referred as MVA-B ΔA41L/ΔB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B ΔA41L/ΔB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4⁺ and CD8⁺ T cells, with the CD8⁺ T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B ΔA41L/ΔB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4⁺ and CD8⁺ T-cell immune responses. HIV-1-specific CD4⁺ T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8⁺ T-cell responses, MVA-B ΔA41L/ΔB16R induced more GPN-specific CD8⁺ T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env.

Conclusions/Significance

These findings revealed that MVA-B and MVA-B ΔA41L/ΔB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.     

B7-H1 Blockade Increases Survival of Dysfunctional CD8+ T Cells and Confers Protection against Leishmania donovani Infections

Experimental visceral leishmaniasis (VL) represents an exquisite model to study CD8⁺ T cell responses in a context of chronic inflammation and antigen persistence, since it is characterized by chronic infection in the spleen and CD8⁺ T cells are required for the development of protective immunity. However, antigen-specific CD8⁺ T cell responses in VL have so far not been studied, due to the absence of any defined Leishmania-specific CD8⁺ T cell epitopes. In this study, transgenic Leishmania donovani parasites expressing ovalbumin were used to characterize the development, function, and fate of Leishmania-specific CD8⁺ T cell responses. Here we show that L. donovani parasites evade CD8⁺ T cell responses by limiting their expansion and inducing functional exhaustion and cell death. Dysfunctional CD8⁺ T cells could be partially rescued by in vivo B7-H1 blockade, which increased CD8⁺ T cell survival but failed to restore cytokine production. Nevertheless, B7-H1 blockade significantly reduced the splenic parasite burden. These findings could be exploited for the design of new strategies for immunotherapeutic interventions against VL.     

Role of PKR and Type I IFNs in Viral Control during Primary and Secondary Infection

Type I interferons (IFNs) are known to mediate viral control, and also promote survival and expansion of virus-specific CD8⁺ T cells. However, it is unclear whether signaling cascades involved in eliciting these diverse cellular effects are also distinct. One of the best-characterized anti-viral signaling mechanisms of Type I IFNs is mediated by the IFN-inducible dsRNA activated protein kinase, PKR. Here, we have investigated the role of PKR and Type I IFNs in regulating viral clearance and CD8⁺ T cell response during primary and secondary viral infections. Our studies demonstrate differential requirement for PKR, in viral control versus elicitation of CD8⁺ T cell responses during primary infection of mice with lymphocytic choriomeningitis virus (LCMV). PKR-deficient mice mounted potent CD8⁺ T cell responses, but failed to effectively control LCMV. The compromised LCMV control in the absence of PKR was multifactorial, and linked to less effective CD8⁺ T cell-mediated viral suppression, enhanced viral replication in cells, and lower steady state expression levels of IFN-responsive genes. Moreover, we show that despite normal expansion of memory CD8⁺ T cells and differentiation into effectors during a secondary response, effective clearance of LCMV but not vaccinia virus required PKR activity in infected cells. In the absence of Type I IFN signaling, secondary effector CD8⁺ T cells were ineffective in controlling both LCMV and vaccinia virus replication in vivo. These findings provide insight into cellular pathways of Type I IFN actions, and highlight the under-appreciated importance of innate immune mechanisms of viral control during secondary infections, despite the accelerated responses of memory CD8⁺ T cells. Additionally, the results presented here have furthered our understanding of the immune correlates of anti-viral protective immunity, which have implications in the rational design of vaccines.     

Alphavirus Replicon Particles Expressing TRP-2 Provide Potent Therapeutic Effect on Melanoma through Activation of Humoral and Cellular Immunity

Background

Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8⁺ T cells. Activation of effector CD8⁺ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.

Methodology/Principal Findings

VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8⁺ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8⁺ T cell effector responses, and depends on signaling through activating Fcγ receptors.

Conclusions/Significance

This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8⁺ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.     

Lymphocytic choriomeningitis virus infection yields overlapping CD4+ and CD8+ T-cell responses

Activation of CD4⁺ T cells helps establish and sustain other immune responses. We have previously shown that responses against a broad set of nine CD4⁺ T-cell epitopes were present in the setting of lymphocytic choriomeningitis virus (LCMV) Armstrong infection in the context of H-2^d. This is quite disparate to the H-2^b setting, where only two epitopes have been identified. We were interested in determining whether a broad set of responses was unique to H-2^d or whether additional CD4⁺ T-cell epitopes could be identified in the setting of the H-2^b background. To pursue this question, we infected C57BL/6 mice with LCMV Armstrong and determined the repertoire of CD4⁺ T-cell responses using overlapping 15-mer peptides corresponding to the LCMV Armstrong sequence. We confirmed positive responses by intracellular cytokine staining and major histocompatibility complex (MHC)-peptide binding assays. A broad repertoire of responses was identified, consisting of six epitopes. These epitopes originate from the nucleoprotein (NP) and glycoprotein (GP). Out of the six newly identified CD4⁺ epitopes, four of them also stimulate CD8⁺ T cells in a statistically significant manner. Furthermore, we assessed these CD4⁺ T-cell responses during the memory phase of LCMV Armstrong infection and after infection with a chronic strain of LCMV and determined that a subset of the responses could be detected under these different conditions. This is the first example of a broad repertoire of shared epitopes between CD4⁺ and CD8⁺ T cells in the context of viral infection. These findings demonstrate that immunodominance is a complex phenomenon in the context of helper responses.     

Visualizing Early Splenic Memory CD8+ T Cells Reactivation against Intracellular Bacteria in the Mouse

Memory CD8⁺ T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m). Memory CD8⁺ T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8⁺ T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs) fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m) as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8⁺ T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8⁺ T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8⁺ T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8⁺ T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8⁺ T produce inflammatory cytokines such as IFN-γ and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8⁺ T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8⁺ T cells provide a local response by secreting effector molecules around infected cells.     

Tumor Immunity against a Simian Virus 40 Oncoprotein Requires CD8+ T Lymphocytes in the Effector Immune Phase

The required activities of CD4⁺ T cells and antibody against the virally encoded oncoprotein simian virus 40 (SV40) Tag have previously been demonstrated by our laboratory to be mediators in achieving antitumor responses and tumor protection through antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we further characterize the necessary immune cell components that lead to systemic tumor immunity within an experimental pulmonary metastatic model as the result of SV40 Tag immunization and antibody production. Immunized animals depleted of CD8⁺ T cells at the onset of experimental tumor cell challenge developed lung tumor foci and had an overall decreased survival due to lung tumor burden, suggesting a role for CD8⁺ T cells in the effector phase of the immune response. Lymphocytes and splenocytes harvested from SV40 Tag-immunized mice experimentally inoculated with tumor cells synthesized increased in vitro levels of the Th1 cytokine gamma interferon (IFN-γ), as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. CD8⁺ T-cell activity was also heightened in SV40 Tag-immunized and tumor cell-challenged mice, based upon intracellular production of perforin, confirming the cytolytic properties of CD8⁺ T cells against tumor cell challenge. Altogether, these data point to the role of recombinant SV40 Tag protein immunization in initiating a cytotoxic T-lymphocyte (CTL) response during tumor cell dissemination and growth. The downstream activity of CD8⁺ T cells within this model is likely initiated from SV40 Tag-specific antibody mediating ADCC tumor cell destruction.Determining the immunologic mechanisms involved in antitumor responses can provide valuable insight into developing and formulating appropriate immunotherapeutic strategies against a range of human cancers (25). Cell-mediated immunity involving CD8⁺ T lymphocytes is generally regarded as the primary response to utilize due to its potent and efficient cytotoxicity against tumor cell targets in vitro and in animal models (10). Indeed, the proof of concept of this approach is best characterized by specialized conditioning protocols that involve autologous transfer of tumor infiltrating lymphocytes (TILs) in metastatic melanoma patients, with objective responses that approximate 70% (8). However, the efficacy of TILs harvested from additional cancer types have been less than effective, and additional strategies, such as genetic modification of peripheral blood mononuclear cells, are being explored to improve and extend the approach of cytotoxic T-lymphocyte (CTL) immunotherapy clinically (33, 46).The roles of immune components such as CD4⁺ T cells and antibody have been given less attention within the context of promoting tumor immunity against a range of tumor antigens. For example, the ability of CD4⁺ T cells to activate humoral immunity can lead to antitumor responses that involve antibody-dependent cell-mediated cytotoxicity (ADCC) (17). In this scenario, antibody binds its targeted antigen and effectors such as natural killer (NK) cells lyse tumorigenic cells through interaction with the Fc region of the bound antibody. The efficacy of ADCC has been realized in scenarios involving breast cancer and non-Hodgkin''s lymphoma, for example, and to date, the only FDA-approved immunologic treatments against these malignancies involve antibody-based therapies (5).The concurrent roles of antibody—with specific emphasis on ADCC—and CD8⁺ T-cell immunity within the context of tumor immunity have not been widely reported. Several recent studies have commented on the ability of antibody-bound tumor cells, particularly as a whole tumor cell-dendritic cell (DC) vaccination approach, to initiate CTL activity by engaging DCs through Fc receptors (9, 19, 34). However, to our knowledge, the mechanistic aspects of ADCC (e.g., NK-mediated lysis) promoting CD8⁺ T-cell activity have been explored in relatively few studies (27, 41). From an immunotherapeutic standpoint, it may be preferable in certain settings to induce both the humoral and cell-mediated arms of the immune system to offset the progression of tumor cell growth and dissemination. Namely, these strategies could include active or passive approaches to first effectively induce ADCC in response to a tumor antigen, which would promote CTL activity against additional tumor targets through cross-presentation.Our laboratory has been involved in determining the immunologic mechanisms of tumor immunity induced by the virally encoded tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag). The mechanistic aspects of SV40 Tag-induced tumor immunity have been examined within an experimental murine model of pulmonary metastasis. To date, CD4⁺ T cells and SV40 Tag-specific antibody have been implicated as required immune components within this murine system in order to achieve complete systemic tumor immunity (18). These studies demonstrated that during the course of immunization with SV40 Tag (i.e., the induction-phase response), CD4⁺ T cells were required to induce an SV40 Tag humoral response. The specific role of the antibody response against an experimental tumor cell challenge was observed to involve ADCC-mediated clearance pathways (4, 23).In the present study, we further characterize the immunologic response to SV40 Tag immunization by observing the necessary immune cell components following experimental challenge (i.e., the effector-phase response) with a tumor cell line expressing SV40 Tag. With the development of an SV40 Tag antibody response following SV40 Tag immunization in vivo, CD8⁺ T-cell depletion during the effector phase resulted in the formation of lung tumor foci and decreased survival not observed with the abrogation of CD4⁺ T cells. SV40 Tag-immunized mice also displayed a heightened Th1 response and CD8⁺ CTL activity after experimental tumor cell challenge, as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. In all, these data indicate that CD8⁺ T cells mediate tumor immunity following antibody activation in response to the tumor-specific antigen SV40 Tag. We hypothesize that CD8⁺ T-cell activity is initiated due to cross-presentation mechanisms as a result of ADCC activity against SV40 Tag. We are not aware of another published report that formulates a role for ADCC activity against a viral oncoprotein in this manner in order to engage CD8⁺ T-cell activation.SV40 Tag has been reported to be expressed in a number of human cancers, including malignant pleural mesothelioma and non-Hodgkin''s lymphoma, although a causal link between SV40 infection and tumorigenesis is uncertain (11, 24, 35). The results of this study have direct implications for the construction of an appropriate immunotherapeutic strategy for patients suffering malignancies expressing the SV40 Tag tumor-specific antigen.     

Tip30 controls differentiation of murine mammary luminal progenitor to estrogen receptor-positive luminal cell through regulating FoxA1 expression

Estrogen receptor-alpha positive (ER⁺) breast cancers comprise the majority of human breast cancers, but molecular mechanisms underlying this subtype of breast cancers remain poorly understood. Here, we show that ER⁺ mammary luminal tumors arising in Tip30^−/−MMTV-Neu mice exhibited increased enrichment of luminal progenitor gene signature. Deletion of the Tip30 gene increased proportion of mammary stem and progenitor cell populations, and raised susceptibility to ER⁺ mammary luminal tumors in female Balb/c mice. Moreover, Tip30^−/− luminal progenitors displayed increases in propensity to differentiate to mature ER⁺ luminal cells and FoxA1 expression. Knockdown of FoxA1 expression in Tip30^−/− progenitors by shRNA specific for FoxA1 reduced their differentiation toward ER⁺ mature luminal cells. Taken together, our results suggest that TIP30 is a key regulator for maintaining ER⁺ and ER⁻luminal pools in the mammary luminal lineage, and loss of it promotes expansion of ER⁺ luminal progenitors and mature cells and ER⁺ mammary tumorigenesis.     

Effector CD4+ T-cell involvement in clearance of infectious herpes simplex virus type 1 from sensory ganglia and spinal cords

In primary infection, CD8⁺ T cells are important for clearance of infectious herpes simplex virus (HSV) from sensory ganglia. In this study, evidence of CD4⁺ T-cell-mediated clearance of infectious HSV type 1 (HSV-1) from neural tissues was also detected. In immunocompetent mice, HSV-specific CD4⁺ T cells were present in sensory ganglia and spinal cords coincident with HSV-1 clearance from these sites and remained detectable at least 8 months postinfection. Neural CD4⁺ T cells isolated at the peak of neural infection secreted gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), or IL-4 after stimulation with HSV antigen. HSV-1 titers in neural tissues were greatly reduced over time in CD8⁺ T-cell-deficient and CD8⁺ T-cell-depleted mice, suggesting that CD4⁺ T cells could mediate clearance of HSV-1 from neural tissue. To examine possible mechanisms by which CD4⁺ T cells resolved neural infection, CD8⁺ T cells were depleted from perforin-deficient or FasL-defective mice. Clearance of infectious virus from neural tissues was not significantly different in perforin-deficient or FasL-defective mice compared to wild-type mice. Further, in spinal cords and brains after vaginal HSV-1 challenge of chimeric mice expressing both perforin and Fas or neither perforin nor Fas, virus titers were significantly lower than in control mice. Thus, perforin and Fas were not required for clearance of infectious virus from neural tissues. These results suggest that HSV-specific CD4⁺ T cells are one component of a long-term immune cell presence in neural tissues following genital HSV-1 infection and play a role in clearance of infectious HSV-1 at neural sites, possibly via a nonlytic mechanism.     

T- and B-Cell-Mediated Protection Induced by Novel,Live Attenuated Pertussis Vaccine in Mice. Cross Protection against Parapertussis

Background

Despite the extensive use of efficacious vaccines, pertussis still ranks among the major causes of childhood mortality worldwide. Two types of pertussis vaccines are currently available, whole-cell, and the more recent acellular vaccines. Because of reduced reactogenicity and comparable efficacy acellular vaccines progressively replace whole-cell vaccines. However, both types require repeated administrations for optimal efficacy. We have recently developed a live attenuated vaccine candidate, named BPZE1, able to protect infant mice after a single nasal administration.

Methodology/Principal Findings

We determined the protective mechanism of BPZE1-mediated immunity by using passive transfer of T cells and antibodies from BPZE1-immunized mice to SCID mice. Clearance of Bordetella pertussis from the lungs was mediated by both BPZE1-induced antibodies and CD4⁺, but not by CD8⁺ T cells. The protective CD4⁺ T cells comprised IFN-γ-producing and IL-17-producing subsets, indicating that BPZE1 induces both Th1 and Th17 CD4⁺ T cells. In addition, and in contrast to acellular pertussis vaccines, BPZE1 also cross-protected against Bordetella parapertussis infection, but in this case only the transfer of CD4⁺ T cells conferred protection. Serum from BPZE1-immunized mice was not able to kill B. parapertussis and did not protect SCID mice against B. parapertussis infection.

Conclusions/Significance

The novel live attenuated pertussis vaccine BPZE1 protects in a pre-clinical mouse model against B. pertussis challenge by both BPZE1-induced antibodies and CD4⁺ T cell responses. It also protects against B. parapertussis infection. However, in this case protection is only T cell mediated.     

Relationship between Regulatory T Cells and Immune Activation in Human Immunodeficiency Virus-Infected Patients Interrupting Antiretroviral Therapy

Persistent immune activation plays a central role in driving Human Immunodeficiency Virus (HIV) disease progression. Whether CD4+CD25+ regulatory T cells (Tregs) are harmful by suppressing HIV-specific immune responses and/or beneficial through a decrease in immune activation remains debatable. We analysed the relationship between proportion and number of regulatory T cells (Tregs) and immune activation in HIV-infected patients interrupting an effective antiretroviral therapy (ART). Twenty-five patients were included in a substudy of a prospective multicenter trial of treatment interruption (TI) (ANRS 116). Proportions and numbers of Tregs and the proportion of activated CD4 and CD8 T cells were assessed at baseline and month 12 (M12) of TI. Specific anti-HIV CD4 and CD8 responses were investigated at baseline and M12. Non parametric univariate analyses and multivariate linear regression models were conducted. At baseline, the proportion of Tregs negatively correlated with the proportion of HLA-DR⁺CD8⁺T cells (r = −0.519). Following TI, the proportion of Tregs increased from 6.3% to 7.2% (p = 0.029); absolute numbers of Tregs decreased. The increase in the proportion of HLA-DR⁺CD38⁺CD8⁺T cells was significantly related to the increase in proportion of Tregs (p = 0.031). At M12, the proportion of Tregs did not negatively correlate with CD8 T-cell activation. Nevertheless, Tregs retain a suppressive function since depletion of Treg-containing CD4⁺CD25⁺ cells led to an increase in lymphoproliferative responses in most patients studied. Our data suggest that Tregs are efficient in controlling residual immune activation in patients with ART-mediated viral suppression. However, the insufficient increase in the proportion and/or the decrease in the absolute number of Tregs result in a failure to control immune activation following TI.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00118677","term_id":"NCT00118677"}}NCT00118677     

Efficient, Repeated Adenovirus-Mediated Gene Transfer in Mice Lacking both Tumor Necrosis Factor Alpha and Lymphotoxin α

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, and the related cytokine lymphotoxin α (LTα), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-α/LTα^−/−), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-α/LTα^−/− and TNF-α/LTα^+/− mice versus TNF-α/LTα^+/+ mice links antibody levels to TNF-α/LTα gene dosage. Due to the absence of neutralizing antibodies, the TNF-α/LTα knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-α in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.     

Clearance of an Influenza A Virus by CD4+ T Cells Is Inefficient in the Absence of B Cells

The primary CD8⁺ T-cell response protected most B-cell-deficient μMT mice against intranasal infection with the HKx31 influenza A virus. Prior exposure did not prevent reinfection upon homologous challenge, and the recall CD8⁺ T-cell response cleared the virus from the lung within 7 days. Depleting the CD8⁺ T cells substantially reduced the capacity of these primed mice to deal with the infection, in spite of evidence for established CD4⁺ T-cell memory. Thus, the control of this relatively mild influenza virus by both primary and secondary CD4⁺ T-cell responses is relatively inefficient in the absence of B cells and CD8⁺ T cells.     

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4⁺ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8⁺ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4⁺CD25^hiFOXP3⁺ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8⁺ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8⁺ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8⁺ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8⁺ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.     

"Negative vaccination" by specific CD4 T cell tolerisation enhances virus-specific protective antibody responses

Background

Cooperation of CD4⁺ T helper cells with specific B cells is crucial for protective vaccination against pathogens by inducing long-lived neutralizing antibody responses. During infection with persistence-prone viruses, prolonged virus replication correlates with low neutralizing antibody responses. We recently described that a viral mutant of lymphocytic choriomeningitis virus (LCMV), which lacks a T helper epitope, counterintuitively induced an enhanced protective antibody response. Likewise, partial depletion of the CD4⁺ T cell compartment by using anti-CD4 antibodies enhanced protective antibodies.

Principal Findings

Here we have developed a protocol to selectively reduce the CD4⁺ T cell response against viral CD4⁺ T cell epitopes. We demonstrate that in vivo treatment with LCMV-derived MHC-II peptides induced non-responsiveness of specific CD4⁺ T cells without affecting CD4⁺ T cell reactivity towards other antigens. This was associated with accelerated virus-specific neutralizing IgG-antibody responses. In contrast to a complete absence of CD4⁺ T cell help, tolerisation did not impair CD8⁺ T cell responses.

Conclusions

This result reveals a novel “negative vaccination” strategy where specific CD4⁺ T cell unresponsiveness may be used to enhance the delayed protective antibody responses in chronic virus infections.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

T lymphocytes contribute to antiviral immunity and pathogenesis in experimental human metapneumovirus infection

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a leading cause of lower respiratory tract infections in children, the elderly, and immunocompromised patients. Virus- and host-specific mechanisms of pathogenesis and immune protection are not fully understood. By an intranasal inoculation model, we show that hMPV-infected BALB/c mice developed clinical disease, including airway obstruction and hyperresponsiveness (AHR), along with histopathologic evidence of lung inflammation and viral replication. hMPV infection protected mice against subsequent viral challenge, as demonstrated by undetectable viral titers, lack of body weight loss, and a significant reduction in the level of lung inflammation. No cross-protection with other paramyxoviruses, such as respiratory syncytial virus, was observed. T-lymphocyte depletion studies showed that CD4⁺ and CD8⁺ T cells cooperate synergistically in hMPV eradication during primary infection, but CD4⁺ more than CD8⁺ T cells also enhanced clinical disease and lung pathology. Concurrent depletion of CD4⁺ and CD8⁺ T cells completely blocked airway obstruction as well as AHR. Despite impaired generation of neutralizing anti-hMPV antibodies in the absence of CD4⁺ T cells, mice had undetectable viral replication after hMPV challenge and were protected from clinical disease, suggesting that protection can be provided by an intact CD8⁺ T-cell compartment. Whether these findings have implications for naturally acquired human infections remains to be determined.     

Correlation of memory T cell responses against TRAP with protection from clinical malaria, and CD4 CD25 high T cells with susceptibility in Kenyans

Background

Immunity to malaria develops naturally in endemic regions, but the protective immune mechanisms are poorly understood. Many vaccination strategies aim to induce T cells against diverse pre-erythrocytic antigens, but correlates of protection in the field have been limited. The objective of this study was to investigate cell-mediated immune correlates of protection in natural malaria. Memory T cells reactive against thrombospondin-related adhesive protein (TRAP) and circumsporozoite (CS) protein, major vaccine candidate antigens, were measured, as were frequencies of CD4⁺ CD25^high T cells, which may suppress immunity, and CD56⁺ NK cells and γδ T cells, which may be effectors or may modulate immunity.

Methodology and Principal Findings

112 healthy volunteers living in rural Kenya were entered in the study. Memory T cells reactive against TRAP and CS were measured using a cultured IFNγ ELISPOT approach, whilst CD4⁺ CD25^high T cells, CD56⁺ NK cells, and γδ T cells were measured by flow cytometry. We found that T cell responses against TRAP were established early in life (<5 years) in contrast to CS, and cultured ELISPOT memory T cell responses did not correlate with ex-vivo IFNγ ELISPOT effector responses. Data was examined for associations with risk of clinical malaria for a period of 300 days. Multivariate logistic analysis incorporating age and CS response showed that cultured memory T cell responses against TRAP were associated with a significantly reduced incidence of malaria (p = 0.028). This was not seen for CS responses. Higher numbers of CD4⁺ CD25^high T cells, potentially regulatory T cells, were associated with a significantly increased risk of clinical malaria (p = 0.039).

Conclusions

These data demonstrate a role for central memory T cells in natural malarial immunity and support current vaccination strategies aimed at inducing durable protective T cell responses against the TRAP antigen. They also suggest that CD4⁺ CD25^high T cells may negatively affect naturally acquired malarial immunity.