Broadly neutralizing humanized SARS-CoV-2 antibody binds to a conserved epitope on Spike and provides antiviral protection through inhalation-based delivery in non-human primates.

The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.

Administration of airborne pathogens in non-human primates.

PURPOSE: Airborne pathogen scan penetrate in human respiratory tract and can cause illness. The use of animal models to predict aerosol deposition and study respiratory disease pathophysiology is therefore important for research and a prerequisite to test and study the mechanism of action of treatment. NHPs are relevant animal species for inhalation studies because of their similarities with humans in terms of anatomical structure, respiratory parameters and immune system. MATERIALS AND METHODS: The aim of this review is to provide an overview of the state of the art of pathogen aerosol studies performed in non-human primates (NHPs). Herein, we present and discuss the deposition of aerosolized bacteria and viruses. In this review, we present important advantages of using NHPs as model for inhalation studies. RESULTS: We demonstrate that deposition in the respiratory tract is not only a function of aerosol size but also the technique of administration influences the biological activity and site of aerosol deposition. Finally, we observe an influence of a region of pathogen deposition in the respiratory tract on the development of the pathophysiological effect in NHPs. CONCLUSION: The wide range of methods used for the delivery of pathogento NHP respiratory airways is associated with varying doses and deposition profiles in the airways.

Comparison of Aerosol Deposition Between a Cynomolgus Macaque and a 3D Printed Cast Model of the Animal.

PURPOSE: Preclinical aerosol studies using animals are essential for evaluating toxic or therapeutic effects on human respiratory tract. Macaques are relevant animal models for respiratory studies, but they are sensitive, expensive and difficult-to-access. METHODS: In the context of preliminary studies before animal experiments, we set up an alternative in vitro anatomical model of macaque airways to reduce, refine and replace (3Rs) the animals. We printed an in vitro anatomical cast until the third bronchial division from X-ray computed tomography data of a healthy cynomolgus macaque. This in vitro model was then connected to a respiratory pump to mimic macaque's breathing. We assessed the relevance of this in vitro model, by comparing aerosol deposition patterns obtained with the anatomical model and in three macaques using planar gamma camera imaging. DTPA-99mTechnetium aerosols were produced using three jet nebulizers, generating three different particle sizes: 13.1, 3.2 and 0.93 µm in terms of the mass median aerodynamic diameter (MMAD). RESULTS: The data showed no statistical differences between the animal and anatomical in vitro models in terms of total aerosol deposited in the airways. However, the distribution of the deposition in the airways showed a higher deposited fraction in the upper respiratory tract in the animals than the in vitro model for all particle sizes. CONCLUSIONS: The anatomical printed model appears to be a relevant in vitro tool to predict total aerosol deposition in macaque airways.

Isolation and phenotypic characterization of human and nonhuman primate intestinal lamina propria mononuclear cells.

Isolation of viable immune cells from tissues is critically important to characterize cellular and molecular processes during homeostasis and disease. Here, we provide an optimized protocol to achieve high yields of viable intestinal lamina propria mononuclear cells (LPMCs). We describe steps for intestinal tissue collection from humans and nonhuman primates, followed by mechanical disruption and enzymatic digestion. Furthermore, we detail characterization of the mononuclear phagocyte (MP) subtypes by flow cytometry analysis. The protocol is repeatable and scalable for downstream applications. For complete details on the use and execution of this protocol, please refer to Cavarelli et al. (2022).

Multi-component prime-boost Chlamydia trachomatis vaccination regimes induce antibody and T cell responses and accelerate clearance of infection in a non-human primate model.

It is of international priority to develop a vaccine against sexually transmitted Chlamydia trachomatis infections to combat the continued global spread of the infection. The optimal immunization strategy still remains to be fully elucidated. The aim of this study was to evaluate immunization strategies in a nonhuman primate (NHP) model. Cynomolgus macaques (Macaqua fascicularis) were immunized following different multi-component prime-boost immunization-schedules and subsequently challenged with C. trachomatis SvD in the lower genital tract. The immunization antigens included the recombinant protein antigen CTH522 adjuvanted with CAF01 or aluminium hydroxide, MOMP DNA antigen and MOMP vector antigens (HuAd5 MOMP and MVA MOMP). All antigen constructs were highly immunogenic raising significant systemic C. trachomatis-specific IgG responses. In particularly the CTH522 protein vaccinated groups raised a fast and strong pecificsIgG in serum. The mapping of specific B cell epitopes within the MOMP showed that all vaccinated groups, recognized epitopes near or within the variable domains (VD) of MOMP, with a consistent VD4 response in all animals. Furthermore, serum from all vaccinated groups were able to in vitro neutralize both SvD, SvE and SvF. Antibody responses were reflected on the vaginal and ocular mucosa, which showed detectable levels of IgG. Vaccines also induced C. trachomatis-specific cell mediated responses, as shown by in vitro stimulation and intracellular cytokine staining of peripheral blood mononuclear cells (PBMCs). In general, the protein (CTH522) vaccinated groups established a multifunctional CD4 T cell response, whereas the DNA and Vector vaccinated groups also established a CD8 T cells response. Following vaginal challenge with C. trachomatis SvD, several of the vaccinated groups showed accelerated clearance of the infection, but especially the DNA group, boosted with CAF01 adjuvanted CTH522 to achieve a balanced CD4/CD8 T cell response combined with an IgG response, showed accelerated clearance of the infection.

Computed tomography and [18F]-FDG PET imaging provide additional readouts for COVID-19 pathogenesis and therapies evaluation in non-human primates.

Non-human primates (NHPs) are particularly relevant as preclinical models for SARS-CoV-2 infection and nuclear imaging may represent a valuable tool for monitoring infection in this species. We investigated the benefit of computed X-ray tomography (CT) and [18F]-FDG positron emission tomography (PET) to monitor the early phase of the disease in a large cohort (n = 76) of SARS-CoV-2 infected macaques. Following infection, animals showed mild COVID-19 symptoms including typical lung lesions. CT scores at the acute phase reflect the heterogeneity of lung burden following infection. Moreover, [18F]-FDG PET revealed that FDG uptake was significantly higher in the lungs, nasal cavities, lung-draining lymph nodes, and spleen of NHPs by 5 days postinfection compared to pre-infection levels, indicating early local inflammation. The comparison of CT and PET data from previous COVID-19 treatments or vaccines we tested in NHP, to this large cohort of untreated animals demonstrated the value of in vivo imaging in preclinical trials.

Antiviral efficacy of favipiravir against Zika and SARS-CoV-2 viruses in non-human primates.

The COVID-19 pandemic has exemplified that rigorous evaluation in large animal models is key for translation from promising in vitro results to successful clinical implementation. Among the drugs that have been largely tested in clinical trials but failed so far to bring clear evidence of clinical efficacy is favipiravir, a nucleoside analogue with large spectrum activity against several RNA viruses in vitro and in small animal models. Here, we evaluate the antiviral activity of favipiravir against Zika or SARS-CoV-2 virus in cynomolgus macaques. In both models, high doses of favipiravir are initiated before infection and viral kinetics are evaluated during 7 to 15 days after infection. Favipiravir leads to a statistically significant reduction in plasma Zika viral load compared to untreated animals. However, favipiravir has no effects on SARS-CoV-2 viral kinetics, and 4 treated animals have to be euthanized due to rapid clinical deterioration, suggesting a potential role of favipiravir in disease worsening in SARS-CoV-2 infected animals. To summarize, favipiravir has an antiviral activity against Zika virus but not against SARS-CoV-2 infection in the cynomolgus macaque model. Our results support the clinical evaluation of favipiravir against Zika virus but they advocate against its use against SARS-CoV-2 infection.

Modelling the response to vaccine in non-human primates to define SARS-CoV-2 mechanistic correlates of protection.

The definition of correlates of protection is critical for the development of next-generation SARS-CoV-2 vaccine platforms. Here, we propose a model-based approach for identifying mechanistic correlates of protection based on mathematical modelling of viral dynamics and data mining of immunological markers. The application to three different studies in non-human primates evaluating SARS-CoV-2 vaccines based on CD40-targeting, two-component spike nanoparticle and mRNA 1273 identifies and quantifies two main mechanisms that are a decrease of rate of cell infection and an increase in clearance of infected cells. Inhibition of RBD binding to ACE2 appears to be a robust mechanistic correlate of protection across the three vaccine platforms although not capturing the whole biological vaccine effect. The model shows that RBD/ACE2 binding inhibition represents a strong mechanism of protection which required significant reduction in blocking potency to effectively compromise the control of viral replication.

Non-human primate models of human respiratory infections.

Respiratory pathogens represent a great burden for humanity and a potential source of new pandemics, as illustrated by the recent emergence of coronavirus disease 2019 (COVID-19). In recent decades, biotechnological advances have led to the development of numerous innovative therapeutic molecules and vaccine immunogens. However, we still lack effective treatments and vaccines against many respiratory pathogens. More than ever, there is a need for a fast, predictive, preclinical pipeline, to keep pace with emerging diseases. Animal models are key for the preclinical development of disease management strategies. The predictive value of these models depends on their ability to reproduce the features of the human disease, the mode of transmission of the infectious agent and the availability of technologies for monitoring infection. This review focuses on the use of non-human primates as relevant preclinical models for the development of prevention and treatment for human respiratory infections.

SARS-CoV-2 infection in nonhuman primates alters the composition and functional activity of the gut microbiota.

The current pandemic of coronavirus disease (COVID) 2019 constitutes a global public health issue. Regarding the emerging importance of the gut-lung axis in viral respiratory infections, analysis of the gut microbiota's composition and functional activity during a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection might be instrumental in understanding and controling COVID 19. We used a nonhuman primate model (the macaque), that recapitulates mild COVID-19 symptoms, to analyze the effects of a SARS-CoV-2 infection on dynamic changes of the gut microbiota. 16S rRNA gene profiling and analysis of ß diversity indicated significant changes in the composition of the gut microbiota with a peak at 10-13 days post-infection (dpi). Analysis of bacterial abundance correlation networks confirmed disruption of the bacterial community at 10-13 dpi. Some alterations in microbiota persisted after the resolution of the infection until day 26. Some changes in the relative bacterial taxon abundance associated with infectious parameters. Interestingly, the relative abundance of Acinetobacter (Proteobacteria) and some genera of the Ruminococcaceae family (Firmicutes) was positively correlated with the presence of SARS-CoV-2 in the upper respiratory tract. Targeted quantitative metabolomics indicated a drop in short-chain fatty acids (SCFAs) and changes in several bile acids and tryptophan metabolites in infected animals. The relative abundance of several taxa known to be SCFA producers (mostly from the Ruminococcaceae family) was negatively correlated with systemic inflammatory markers while the opposite correlation was seen with several members of the genus Streptococcus. Collectively, SARS-CoV-2 infection in a nonhuman primate is associated with changes in the gut microbiota's composition and functional activity.

Primary infection with simian immunodeficiency virus: plasmacytoid dendritic cell homing to lymph nodes, type I interferon, and immune suppression.

Plasmacytoid dendritic cells (pDCs) are antigen-presenting cells that develop into type-I interferon (IFN-I)-producing cells in response to pathogens. Their role in human immunodeficiency virus (HIV) pathogenesis needs to be understood. We analyzed their dynamics in relation to innate and adaptive immunity very early during the acute phase of simian immunodeficiency virus (SIV) infection in 18 macaques. pDC counts decreased in blood and increased in peripheral lymph nodes, consistent with early recruitment in secondary lymphoid tissues. These changes correlated with the kinetic and intensity of viremia and were associated with a peak of plasma IFN-I. IFN-I and viremia were positively correlated with functional activity of the immune suppression associated enzyme indoleamine-2,3-dioxygenase (IDO) and FoxP3(+)CD8(+) T cells, which both negatively correlated with SIV-specific T-cell proliferation and CD4(+) T-cell activation. These data suggest that pDCs and IFN-I play a key role in shaping innate and adaptive immunity toward suppressive pathways during the acute phase of SIV/HIV primary infection.

Innate and Adaptive Anti-SIV Responses in Macaque Semen: Implications for Infectivity and Risk of Transmission.

HIV-1 infection is transmitted primarily by sexual exposure, with semen being the principal contaminated fluid. However, HIV-specific immune response in semen has been understudied. We investigated specific parameters of the innate, cellular, and humoral immune response that may affect semen infectivity in macaques infected with SIVmac251. Serial semen levels of cytokines and chemokines, SIV-specific antibodies, neutralization, and FcγR-mediated functions and SIV-specific T-cell responses were assessed and compared to systemic responses across 53 cynomolgus macaques. SIV infection induced an overall inflammatory state in the semen. Several pro-inflammatory molecules correlated with SIV virus levels. Effector CD8+ T cells were expanded in semen upon infection. SIV-specific CD8+ T-cells that expressed multiple effector molecules (IFN-γ+MIP-1ß+TNF+/-) were induced in the semen of a subset of SIV-infected macaques, but this did not correlate with local viral control. SIV-specific IgG, commonly capable of engaging the FcγRIIIa receptor, was detected in most semen samples although this positively correlated with seminal viral load. Several inflammatory immune responses in semen develop in the context of higher levels of SIV seminal plasma viremia. These inflammatory immune responses could play a role in viral transmission and should be considered in the development of preventive and prophylactic vaccines.

Capsid-specific removal of circulating antibodies to adeno-associated virus vectors.

Neutralizing antibodies directed against adeno-associated virus (AAV) are commonly found in humans. In seropositive subjects, vector administration is not feasible as antibodies neutralize AAV vectors even at low titers. Consequently, a relatively large proportion of humans is excluded from enrollment in clinical trials and, similarly, vector redosing is not feasible because of development of high-titer antibodies following AAV vector administration. Plasmapheresis has been proposed as strategy to remove anti-AAV antibodies from the bloodstream. Although safe and relatively effective, the technology has some limitations mainly related to the nonspecific removal of all circulating IgG. Here we developed an AAV-specific plasmapheresis column which was shown to efficiently and selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma. We showed the nearly complete removal of anti-AAV antibodies from high titer purified human IgG pools and plasma samples, decreasing titers to levels that allow AAV vector administration in mice. These results provide proof-of-concept of a method for the AAV-specific depletion of neutralizing antibodies in the setting of in vivo gene transfer.

Dynamics of viral replication in blood and lymphoid tissues during SIVmac251 infection of macaques.

BACKGROUND: Extensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied. RESULTS: The evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT), with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected. CONCLUSION: We demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important implications for the targeting of HIV treatment to these diverse compartments.

Effect of SIVmac infection on plasmacytoid and CD1c+ myeloid dendritic cells in cynomolgus macaques.

Dendritic cells (DCs) are known to be essential for the induction and regulation of immune responses. Non-human primates are essential in biomedical research and contribute to our understanding of the involvement of DCs in human infectious diseases. However, no direct single-platform method for quantifying DC precursors has yet been optimized in macaques to give accurate absolute blood counts of these rare-event cell populations in the blood. We adapted a rapid whole-blood assay for the absolute quantification of DCs in cynomolgus macaques by four-colour flow cytometry, using a single-platform assay compatible with human blood. Cynomolgus macaque plasmacytoid DCs (pDCs) and CD1c(+) myeloid DCs (CD1c(+) mDCs) were quantified in the blood of 34 healthy macaques and the results obtained were compared with those for blood samples from 11 healthy humans. In addition, circulating absolute numbers of pDCs were quantified in cynomolgus macaques chronically infected with SIVmac. During infection, pDC counts decreased whereas circulating CD1c(+) mDC counts increased. Information regarding absolute pDC and mDC counts in non-human primates may improve our understanding of the role of these cells in SIV/HIV infection and in other infectious diseases.

Reconstitution of the myeloid and lymphoid compartments after the transplantation of autologous and genetically modified CD34+ bone marrow cells, following gamma irradiation in cynomolgus macaques.

BACKGROUND: Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques. RESULTS: The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% +/- 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% +/- 10%). A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery. CONCLUSION: The transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T- and B-lymphocyte, thrombocyte and red blood cell compartments.

FoxP3+ CD25+ CD8+ T-cell induction during primary simian immunodeficiency virus infection in cynomolgus macaques correlates with low CD4+ T-cell activation and high viral load.

The early immune response fails to prevent the establishment of chronic human immunodeficiency virus (HIV) infection but may influence viremia during primary infection, thereby possibly affecting long-term disease progression. CD25(+) FoxP3(+) regulatory T cells may contribute to HIV/simian immunodeficiency virus (SIV) pathogenesis by suppressing efficient antiviral responses during primary infection, favoring high levels of viral replication and the establishment of chronic infection. In contrast, they may decrease immune activation during chronic infection. CD4(+) regulatory T cells have been studied in the most detail, but CD8(+) CD25(+) FoxP3(+) T cells also have regulatory properties. We monitored the dynamics of CD25(+) FoxP3(+) T cells during primary and chronic SIVmac251 infection in cynomolgus macaques. The number of peripheral CD4(+) CD25(+) FoxP3(+) T cells paralleled that of memory CD4(+) T cells, with a rapid decline during primary infection followed by a rebound to levels just below baseline and gradual depletion during the course of infection. No change in the proportion of CD25(+) FoxP3(+) T cells was observed in peripheral lymph nodes. A small number of CD4(+) CD25(+) FoxP3(+) T cells at set point was associated with a high plasma viral load. In contrast, peripheral CD8(+) CD25(+) FoxP3(+) T cells were induced a few days after peak plasma viral load during primary infection. The number of these cells was positively correlated with viral load and negatively correlated with CD4(+) T-cell activation, SIV antigen-specific proliferative responses during primary infection, and plasma viral load at set point, with large numbers of CD8(+) CD25(+) FoxP3(+) T cells being indicative of a poor prognosis.

Dynamics of T-cell responses and memory T cells during primary simian immunodeficiency virus infection in cynomolgus macaques.

Cellular immune responses make an important contribution to both the control of human immunodeficiency virus (HIV) replication and disease progression. We used a pathogenic model of SIVmac251 infection of cynomolgus macaques to longitudinally evaluate cellular immune responses in association with various rates of disease progression. We found an inverse relationship between plasma viral load and the simian immunodeficiency virus (SIV)-specific T cells responses in peripheral blood and lymph nodes. SIV-specific T-cell responses in peripheral blood were transient during primary infection, with the highest responses detected around 3 months after infection. There was also a transient increase of central memory CD8(+) T cells in peripheral blood during primary infection, and effector memory T-cell counts in peripheral lymph nodes were increased. This study emphasizes the importance of the early virus-specific immune responses in the outcome of HIV/SIV disease and provides details about the changes of virus-specific immune responses over time.

GFP-transduced CD34+ and Lin- CD34- hematopoietic stem cells did not adopt a cardiac phenotype in a nonhuman primate model of myocardial infarct.

OBJECTIVE: Studies in mice have reported contradictory results on the contribution of bone marrow cells to myocardial regeneration. This study aims to evaluate their ability to differentiate into cells of cardiac lineage in a nonhuman primate mode of myocardial infarct. MATERIALS AND METHODS: Lin(-)CD34(-) and CD34(+)-enriched bone marrow cells or mobilized peripheral blood cells were transduced with green fluorescent protein (GFP) and injected directly into ischemic myocardium. The fate of the transplanted cells was evaluated using quantitative reverse transcription polymerase chain reaction (QRT-PCR) and immunohistology. Animals were followed-up using echocardiography. RESULTS: QRT-PCR analysis detected from 3% to 10% of the original number of administered GFP(+) cells after 7 days. These GFP(+) cells did not express cardiac tissue-specific markers, but were immunophenotypically consistent with undifferentiated hematopoietic cells. The local production of vascular endothelial growth factor, measured by QRT-PCR, was approximately doubled as compared to the untreated infarcted control heart. Three months after hematopoietic stem cell (HSC) administration, no GFP(+) cells were detected and no evidence of regeneration of the infarcted region was found by histological examination. In contrast, a high level of matrix metalloproteinase 2 was measured in infarct and peri-infarct area. At this time, an improved ejection fraction and decreased left ventricular chamber dimension, which might be also related to a natural course after reperfusion, were observed. CONCLUSIONS: Our data show that GFP(+) CD34(+) and Lin(-)CD34(-)-enriched HSC do not differentiate into cardiomyocytes or into endothelial cells in the infarcted myocardium and that the local production of some growth factors had no positive effect on myocardial regeneration after 3 months.

Loss of reactivity of vaccine-induced CD4 T cells in immunized monkeys after SIV/HIV challenge.

BACKGROUND: Immunization protocols involving priming with DNA and boosting with recombinant live virus vectors such as recombinant modified Vaccinia Ankara (rMVA) are considered as vaccine candidates against HIV. Such protocols improve the outcome of simian/human immunodeficiency virus (SHIV) pathogenic challenge in Rhesus monkeys. OBJECTIVES: To investigate the fate of vaccine-induced T cells after a mucosal SHIV challenge. METHODS: We immunized Rhesus monkeys (Macaca mulatta) by DNA priming followed by rMVA boost. After intrarectal challenge with SHIV 89.6P, immunized animals demonstrated early control of viral replication and stable CD4 T-cell counts. We monitored T-cell responses by measuring IFN-gamma secretion and proliferation. RESULTS: Immunization induced strong and sustained SHIV-specific CD4 and CD8 T-cell responses. CD8 T-cell responses were recalled during acute infection, whereas none of the vaccine-induced SHIV-specific CD4 T-cell responses were recalled. Moreover, most of the CD4 T-cell responses became undetectable in peripheral blood or lymph nodes even after in-vitro peptide stimulation. In contrast, we persistently detected CD4 T-cell responses specific for control recall antigens in infected animals. CONCLUSION: SHIV 89.6P challenge results in a lack of reactivity of vaccine-induced SHIV-specific CD4 T cells. These results may have important implications in the AIDS vaccine field, especially for the evaluation of new vaccine candidates, both in preventive and therapeutic trials.