Immunosuppression Profile of CFZ533 (Iscalimab), a Non-Depleting Anti-CD40 Antibody, and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study.

CFZ533 (iscalimab) is a nondepleting anti-CD40 antibody intended for inhibition of transplant organ rejection and treatment of autoimmune diseases. In a safety assessment in rhesus monkeys, CFZ533 was administered for 13 weeks up to 150 mg/kg/week subcutaneously. CFZ533 was shown previously to completely inhibit primary and secondary T-cell-dependent antibody responses. CD40 is expressed on B cells, antigen-presenting cells, and endothelial and epithelial cells, but is not expressed on T cells. Here, we demonstrate the complete suppression of germinal center formation in lymphoid organs. CFZ533 was well tolerated and did not cause any dose-limiting toxicity. However, the histological evaluation revealed increased numbers of CD4+ and CD8+ T cells in the T-cell-rich areas of lymph nodes enlarged in response to observed adenovirus and Cryptosporidium infections which suggest that T-cell immune function was unaffected. Background infections appear as the condition leading to unraveling the differential immunosuppressive effects by CFZ533. The presence of T cells at lymph nodes draining sites of infections corroborates the immunosuppressive mechanism, which is different from calcineurin-inhibiting drugs. Furthermore, CFZ533 did not show any hematological or microscopic evidence of thromboembolic events in rhesus monkeys, which were previously shown to respond with thromboembolism to treatment with anti-CD154 antibodies.

Vaccine protection against acquisition of neutralization-resistant SIV challenges in rhesus monkeys.

Preclinical studies of human immunodeficiency virus type 1 (HIV-1) vaccine candidates have typically shown post-infection virological control, but protection against acquisition of infection has previously only been reported against neutralization-sensitive virus challenges. Here we demonstrate vaccine protection against acquisition of fully heterologous, neutralization-resistant simian immunodeficiency virus (SIV) challenges in rhesus monkeys. Adenovirus/poxvirus and adenovirus/adenovirus-vector-based vaccines expressing SIV(SME543) Gag, Pol and Env antigens resulted in an 80% or greater reduction in the per-exposure probability of infection against repetitive, intrarectal SIV(MAC251) challenges in rhesus monkeys. Protection against acquisition of infection showed distinct immunological correlates compared with post-infection virological control and required the inclusion of Env in the vaccine regimen. These data demonstrate the proof-of-concept that optimized HIV-1 vaccine candidates can block acquisition of stringent, heterologous, neutralization-resistant virus challenges in rhesus monkeys.

Limited dissemination and shedding of the UL128 complex-intact, UL/b'-defective rhesus cytomegalovirus strain 180.92.

UNLABELLED: The UL128 complex of human cytomegalovirus (CMV) is a major determinant of viral entry into epithelial and endothelial cells and a target for vaccine development. The UL/b' region of rhesus CMV contains several open reading frames, including orthologs of the UL128 complex. We recently showed that the coding content of the rhesus CMV (RhCMV) UL/b' region predicts acute endothelial tropism and long-term shedding in vivo in the rhesus macaque model of CMV infection. The laboratory-passaged RhCMV 180.92 strain has a truncated UL/b' region but an intact UL128 complex. To investigate whether the presence of the UL128 complex alone was sufficient to confer endothelial and epithelial tropism in vivo, we investigated tissue dissemination and viral excretion following experimental RhCMV 180.92 inoculation of RhCMV-seronegative rhesus macaques. We show the presence of at least two virus variants in the RhCMV 180.92 infectious virus stock. A rare variant noted for a nontruncated wild-type-virus-like UL/b' region, rapidly emerged during in vivo replication and showed high-level replication in blood and tissues and excretion in urine and saliva, features similar to those previously reported in naturally occurring wild-type RhCMV infection. In contrast, the predominant truncated version of RhCMV 180.92 showed significantly lower plasma DNAemia and limited tissue dissemination and viral shedding. These data demonstrate that the truncated RhCMV 180.92 variant is attenuated in vivo and suggest that additional UL/b' genes, besides the UL128 complex, are required for optimal in vivo CMV replication and dissemination. IMPORTANCE: An effective vaccine against human CMV infection will need to target genes that are essential for virus propagation and transmission. The human CMV UL128 complex represents one such candidate antigen since it is essential for endothelial and epithelial cell tropism, and is a target for neutralizing antibodies in CMV-infected individuals. In this study, we used the rhesus macaque animal model of CMV infection to investigate the in vivo function of the UL128 complex. Using experimental infection of rhesus macaques with a rhesus CMV virus variant that contained an intact UL128 complex but was missing several other genes, we show that the presence of the UL128 complex alone is not sufficient for widespread tissue dissemination and virus excretion. These data highlight the importance of in vivo studies in evaluating human CMV gene function and suggest that additional UL/b' genes are required for optimal CMV dissemination and transmission.

Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys.

A recombinant adenovirus serotype 5 (rAd5) vector-based vaccine for HIV-1 has recently failed in a phase 2b efficacy study in humans. Consistent with these results, preclinical studies have demonstrated that rAd5 vectors expressing simian immunodeficiency virus (SIV) Gag failed to reduce peak or setpoint viral loads after SIV challenge of rhesus monkeys (Macaca mulatta) that lacked the protective MHC class I allele Mamu-A*01 (ref. 3). Here we show that an improved T-cell-based vaccine regimen using two serologically distinct adenovirus vectors afforded substantially improved protective efficacy in this challenge model. In particular, a heterologous rAd26 prime/rAd5 boost vaccine regimen expressing SIV Gag elicited cellular immune responses with augmented magnitude, breadth and polyfunctionality as compared with the homologous rAd5 regimen. After SIV(MAC251) challenge, monkeys vaccinated with the rAd26/rAd5 regimen showed a 1.4 log reduction of peak and a 2.4 log reduction of setpoint viral loads as well as decreased AIDS-related mortality as compared with control animals. These data demonstrate that durable partial immune control of a pathogenic SIV challenge for more than 500 days can be achieved by a T-cell-based vaccine in Mamu-A*01-negative rhesus monkeys in the absence of a homologous Env antigen. These findings have important implications for the development of next-generation T-cell-based vaccine candidates for HIV-1.

ADCC develops over time during persistent infection with live-attenuated SIV and is associated with complete protection against SIV(mac)251 challenge.

Live-attenuated strains of simian immunodeficiency virus (SIV) routinely confer apparent sterilizing immunity against pathogenic SIV challenge in rhesus macaques. Understanding the mechanisms of protection by live-attenuated SIV may provide important insights into the immune responses needed for protection against HIV-1. Here we investigated the development of antibodies that are functional against neutralization-resistant SIV challenge strains, and tested the hypothesis that these antibodies are associated with protection. In the absence of detectable neutralizing antibodies, Env-specific antibody-dependent cell-mediated cytotoxicity (ADCC) emerged by three weeks after inoculation with SIVΔnef, increased progressively over time, and was proportional to SIVΔnef replication. Persistent infection with SIVΔnef elicited significantly higher ADCC titers than immunization with a non-persistent SIV strain that is limited to a single cycle of infection. ADCC titers were higher against viruses matched to the vaccine strain in Env, but were measurable against viruses expressing heterologous Env proteins. In two separate experiments, which took advantage of either the strain-specificity or the time-dependent maturation of immunity to overcome complete protection against SIV(mac)251 challenge, measures of ADCC activity were higher among the SIVΔnef-inoculated macaques that remained uninfected than among those that became infected. These observations show that features of the antibody response elicited by SIVΔnef are consistent with hallmarks of protection by live-attenuated SIV, and reveal an association between Env-specific antibodies that direct ADCC and apparent sterilizing protection by SIVΔnef.

Frequent infection of neurons by SV40 virus in SIV-infected macaque monkeys with progressive multifocal leukoencephalopathy and meningoencephalitis.

Simian virus 40 (SV40), family Polyomaviridae, in immunocompromised macaques can cause fatal demyelinating central nervous system disease analogous to progressive multifocal leukoencephalopathy caused by John Cunningham (JC) virus in immunocompromised humans. Recently, we have demonstrated that JC virus can infect cerebellar granule cell neurons and cortical pyramidal neurons in immunosuppressed people. To examine whether SV40 neuronal infection occurs spontaneously in immunosuppressed macaques, we analyzed archival brain specimens from 20 simian immunodeficiency virus-infected rhesus with AIDS and 1 cynomolgus post-transplant selected with SV40 brain infection from archival records from 1991 to 2012. In addition to white matter SV40 distribution in classic demyelinating progressive multifocal leukoencephalopathy, some of the 21 monkeys exhibited meningeal, subpial neocortical, and periventricular virus. This distribution pattern corresponded to broader viral tropism with neuronal infection in 14 (66.7%) of 21 cases. In all 14 cases, identified neurons were positive for early SV40 transcript large T antigen, but only 4 of the 14 cases exhibited late viral transcript viral protein 1-positive neurons. SV40-infected neurons were detected in frontal, parietal, occipital, and temporal cortices, hippocampus, thalamus, and brain stem. These observations confirm that spontaneous SV40 neuronal infection occurs in immunosuppressed macaques, which parallels JC virus-neuronal infection in immunosuppressed patients. Neuronal infection may be an important aspect of both SV40 and JC virus neuropathogenesis in their respective hosts.

SIRPα/CD172a and FHOD1 are unique markers of littoral cells, a recently evolved major cell population of red pulp of human spleen.

Asplenic individuals are compromised not only in their ability to destroy infectious agents, but are at increased risk for death from autoimmune disease, certain tumors, and ischemic heart disease. Enhanced mortality is attributed to lack of phagocytes sequestered in spleen that efficiently engulf and destroy appropriate targets, although related cells are found elsewhere. To determine whether a unique population regulates RBC-pathogen clearance and filtration of altered self, we reviewed the anatomic literature and analyzed in situ by immunohistochemistry and immunofluorescence the expression patterns of a little-characterized cell that dominates the splenic red pulp of humans and closely related primates: the venous sinus-lining or littoral cell (LC). High expression of the formin homology domain protein 1 outlines the LC population. Although LCs are endothelial-like in distribution, they express several macrophage-directed proteins, the RBC Duffy Ag receptor for chemokines and T cell coreceptor CD8α/α, yet they lack lineage-associated markers CD34 and CD45. Strikingly, SIRPα (CD172a) expression in human spleen concentrates on LCs, consistent with recent demonstration of a key role in RBC turnover and elimination versus release of infected or altered self. Our results indicate human LCs (SIRPα(+), formin homology domain protein 1(+), CD8α/α(+), CD34(-), CD45(-)) comprise a highly plastic barrier cell population that emerged late in primate evolution coordinate with CD8 expression. Unique to Hominidae, LCs may be the ultimate determinant of which cells recirculate after passage through human spleen.

Patterns of acute rhesus cytomegalovirus (RhCMV) infection predict long-term RhCMV infection.

We previously reported that long-term rhesus cytomegalovirus (RhCMV) excretion in infected macaques was related to UL/b' coding content. Acute biopsy specimens of the inoculation sites from the previous study have now been analyzed to determine whether there were acute phenotypic predictors of long-term RhCMV infection. Only in animals displaying acute endothelial tropism and neutrophilic inflammation was RhCMV excretion detected. The results imply that vaccinating against these early viral determinants would significantly impede long-term RhCMV infection.

Long-term control of simian immunodeficiency virus mac251 viremia to undetectable levels in half of infected female rhesus macaques nasally vaccinated with simian immunodeficiency virus DNA/recombinant modified vaccinia virus Ankara.

The efficacy of two SIV DNA plus recombinant modified vaccinia virus Ankara nasal vaccine regimens, one combined with plasmids expressing IL-2 and IL-15, the other with plasmids expressing GM-CSF, IL-12, and TNF-α, which may better stimulate humoral responses, was evaluated in two female rhesus macaque groups. Vaccination stimulated significant SIV-specific mucosal and systemic cell-mediated immunity in both groups, whereas SIV-specific IgA titers were sporadic and IgG titers negative. All vaccinated animals, except one, became infected after intravaginal SIV(mac251) low-dose challenge. Half of the vaccinated, infected animals (7/13) promptly controlled virus replication to undetectable viremia for the duration of the trial (130 wk) and displayed virological and immunological phenotypes similar to those of exposed, uninfected individuals. When all vaccinated animals were considered, a 3-log viremia reduction was observed, compared with controls. The excellent viral replication containment achieved in vaccinated animals translated into significant preservation of circulating α4ß7(high+)/CD4(+) T cells and of circulating and mucosal CD4(+)/C(M) T cells and in reduced immune activation. A more significant long-term survival was also observed in these animals. Median survival was 72 wk for the control group, whereas >50% of the vaccinated animals were still disease free 130 wk postchallenge, when the trial was closed. There was a statistically significant correlation between levels of CD4(+)/IFN-γ(+) and CD8(+)/IFN-γ(+) T cell percentages on the day of challenge and the control of viremia at week 60 postchallenge or survival. Postchallenge immunological correlates of protection were systemic anti-SIV Gag + Env CD4(+)/IL-2(+), CD4(+)/IFN-γ(+), and CD8(+)/TNF-α(+) T cells and vaginal anti-SIV Gag + Env CD8(+) T cell total monofunctional responses.

Anti-BCMA/CD19 CAR T Cells with Early Immunomodulatory Maintenance for Multiple Myeloma Responding to Initial or Later-Line Therapy.

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)-negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment. SIGNIFICANCE: CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy. This article is highlighted in the In This Issue feature, p. 101.

Quantitative molecular assessment of chimerism across tissues in marmosets and tamarins.

BACKGROUND: Marmosets are playing an increasingly large and important role in biomedical research. They share genetic, anatomical, and physiological similarities with humans and other primate model species, but their smaller sizes, reproductive efficiency, and amenability to genetic manipulation offer an added practicality. While their unique biology can be exploited to provide insights into disease and function, it is also important that researchers are aware of the differences that exist between marmosets and other species. The New World monkey family Callitrichidae, containing both marmoset and tamarin species, typically produces dizygotic twins that show chimerism in the blood and other cells from the hematopoietic lineage. Recently, a study extended these findings to identify chimerism in many tissues, including somatic tissues from other lineages and germ cells. This has raised the intriguing possibility that chimerism may play an increasingly pervasive role in marmoset biology, ranging from natural behavioral implications to increased variability and complexity in biomedical studies. RESULTS: Using a quantitative PCR based methodology, Y-chromosomes can be reliably detected in the females with male fraternal twins allowing for a relative quantification of chimerism levels between individuals and tissues. With this approach in common marmosets (Callithrix jacchus) and cotton-top tamarins (Saguinus oedipus), chimerism was detected across a broad array of tissues. Chimerism levels were significantly higher in tissues primarily derived from the hematopoietic lineage, while they were lower, though still detectable, in tissues with other origins. Interestingly, animals with a characteristic marmoset wasting disease show higher levels of chimerism in those tissues affected. Fibroblast cell lines from chimeric individuals, however, are not found to be chimeric themselves. CONCLUSION: Taken together, the levels of chimerism in tissues of different origins coupled with other lines of evidence suggest that indeed only hematopoietic cell lineages are chimeric in callitrichids. The chimerism detected in other tissues is likely the result of blood or lymphocytic infiltration. Using molecular methods to detect chimerism in a tissue sample seems to have allowed a substantial increase in the ability to detect these minor cell populations.

Durable mucosal simian immunodeficiency virus-specific effector memory T lymphocyte responses elicited by recombinant adenovirus vectors in rhesus monkeys.

The induction of potent and durable cellular immune responses in both peripheral and mucosal tissues may be important for the development of effective vaccines against human immunodeficiency virus type 1 and other pathogens. In particular, effector responses at mucosal surfaces may be critical to respond rapidly to incoming mucosal pathogens. Here we report that intramuscular injection of nonreplicating recombinant adenovirus (rAd) vectors into rhesus monkeys induced remarkably durable simian immunodeficiency virus (SIV)-specific T lymphocyte responses that persisted for over 2 years in both peripheral blood and multiple mucosal tissues, including colorectal, duodenal, and vaginal biopsy specimens, as well as bronchoalveolar lavage fluid. In peripheral blood, SIV-specific T lymphocytes underwent the expected phenotypic evolution from effector memory T cells (T(EM)) to central memory T cells (TCM) following vaccination. In contrast, mucosal SIV-specific T lymphocytes exhibited a persistent and durable T(EM) phenotype that did not evolve over time. These data demonstrate that nonreplicating rAd vectors induce durable and widely distributed effector memory mucosal T lymphocyte responses that are phenotypically distinct from peripheral T lymphocyte responses. Vaccine-elicited T(EM) responses at mucosal surfaces may prove critical for affording protection against invading pathogens at the mucosal portals of entry.

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus.

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.

Immunophenotypic alterations in resident immune cells and myocardial fibrosis in the aging rhesus macaque (Macaca mulatta) heart.

The rhesus macaque (Macaca mulatta) is used extensively in translational biomedical research and drug development studies and is an important model of aging. Macaques often develop myocardial fibrosis with age, which can result in the loss of normal cardiac architecture with the expansion of the extracellular matrix and deposition of collagen. The etiology and pathogenesis of this pernicious process is poorly understood. Cardiac fibrosis was assessed using histologic and immunohistochemical techniques in cardiac tissue sections from 34 rhesus macaques. Overall left ventricular and left ventricular mid-myocardial interstitial/perivascular fibrosis were positively correlated with age (r = .6522, p < .0001 and r = .4704, p = .005, respectively). When divided into young (mean = 2.8 years), middle-aged (mean = 17.5 years), and advanced age (mean = 29.2 years) groups, immunophenotypic characterization of antigen presenting cells revealed differential expression of CD163 and DC-SIGN between the young and middle-aged groups compared to the advanced age group (p < .0001). HAM-56 expression decreased significantly in the advanced age cohort (p = .0021). The expression of CD8, CD163, and DC-SIGN correlated positively with age (r = .3999, p = .0191; r = .5676, p = .0005; r = .5245, p = .0014, respectively). These results show the importance of myocardial fibrosis as a common age-related pathology and additionally, alterations in T cell, macrophage, and dendritic cell phenotype in rhesus macaque myocardium are associated with age but unassociated with the fibrosis.

Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity.

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens have proved highly immunogenic in preclinical studies but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world. Here we show that rAd5 vectors can be engineered to circumvent anti-Ad5 immunity. We constructed novel chimaeric rAd5 vectors in which the seven short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing simian immunodeficiency virus Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs. Moreover, these studies show that recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. Such chimaeric viral vectors may have important practical implications for vaccination and gene therapy.

Mucosal trafficking of vector-specific CD4+ T lymphocytes following vaccination of rhesus monkeys with adenovirus serotype 5.

Post hoc analysis of the phase 2b Step study evaluating a recombinant adenovirus serotype 5 (rAd5)-based HIV-1 vaccine candidate suggested a potential increased risk of HIV-1 acquisition in subjects who were baseline Ad5 seropositive and uncircumcised. These concerns had a profound impact on the HIV-1 vaccine development field, although the mechanism underlying this observation remains unknown. It has been hypothesized that rAd5 vaccination of baseline Ad5-seropositive individuals may have resulted in anamnestic, vector-specific CD4(+) T lymphocytes that could have trafficked to mucosal sites and served as increased targets for HIV-1 infection. Here we show that Ad5-specific CD4(+) T lymphocyte responses at mucosal sites following rAd5-Gag/Pol/Nef vaccination were comparable in rhesus monkeys with and without baseline Ad5 immunity. Moreover, the total cellular inflammatory infiltrates and the CD3(+), CD4(+), HLA-DR(+), Ki67(+), and langerin(+) cellular subpopulations in colorectal and foreskin mucosa were similar in both groups. Thus, no greater trafficking of Ad5-specific CD4(+) T lymphocytes to mucosal target sites was observed following rAd5 vaccination of rhesus monkeys with baseline Ad5 immunity. These findings from this nonhuman primate model provide evidence against the hypothesis that recruitment of vector-specific target cells to mucosal sites led to increased HIV-1 acquisition in Ad5-seropositive, uncircumcised vaccinees in the Step study.

Low-dose mucosal simian immunodeficiency virus infection restricts early replication kinetics and transmitted virus variants in rhesus monkeys.

Defining the earliest virologic events following human immunodeficiency virus type 1 (HIV-1) transmission may be critical for the design of vaccine strategies aimed at blocking acquisition of HIV-1 infection. In particular, the length of the eclipse phase and the number of transmitted virus variants may define the window in which a prophylactic vaccine must act. Here we show that the dose of the virus inoculum affects these key virologic parameters following intrarectal simian immunodeficiency virus (SIV) infection of rhesus monkeys. Low-dose SIV infection resulted in a lengthened eclipse phase, fewer transmitted virus variants, and decreased innate immune activation compared with these parameters in high-dose SIV infection. These data suggest a mechanism by which it may be considerably easier for a vaccine to protect against low-risk HIV-1 transmission than against high-risk HIV-1 transmission. These findings have implications for the design and interpretation of HIV-1 vaccine efficacy studies.

Envelope-modified single-cycle simian immunodeficiency virus selectively enhances antibody responses and partially protects against repeated, low-dose vaginal challenge.

Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV(mac)239 or SIV(mac)251(UCD), neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV(mac)251(UCD), six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.

Immunization with single-cycle SIV significantly reduces viral loads after an intravenous challenge with SIV(mac)239.

Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIV(mac)239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4(+) T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIV(mac)239. However, significantly lower viral loads and higher memory CD4(+) T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIV(mac)239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV.

Non-human primate model of Kaposi's sarcoma-associated herpesvirus infection.

Since Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) was first identified in Kaposi's sarcoma (KS) lesions of HIV-infected individuals with AIDS, the basic biological understanding of KSHV has progressed remarkably. However, the absence of a proper animal model for KSHV continues to impede direct in vivo studies of viral replication, persistence, and pathogenesis. In response to this need for an animal model of KSHV infection, we have explored whether common marmosets can be experimentally infected with human KSHV. Here, we report the successful zoonotic transmission of KSHV into common marmosets (Callithrix jacchus, Cj), a New World primate. Marmosets infected with recombinant KSHV rapidly seroconverted and maintained a vigorous anti-KSHV antibody response. KSHV DNA and latent nuclear antigen (LANA) were readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of infected marmosets. Remarkably, one orally infected marmoset developed a KS-like skin lesion with the characteristic infiltration of leukocytes by spindle cells positive for KSHV DNA and proteins. These results demonstrate that human KSHV infects common marmosets, establishes an efficient persistent infection, and occasionally leads to a KS-like skin lesion. This is the first animal model to significantly elaborate the important aspects of KSHV infection in humans and will aid in the future design of vaccines against KSHV and anti-viral therapies targeting KSHV coinfected tumor cells.