Measles virus (MeV) is an immunosuppressive, extremely contagious RNA virus that remains one of the leading causes of death among children. Wild type MeV strains spread first in lymphatic tissues, causing immunosuppression, and then in epithelial cells of the upper airways, favoring extremely efficient transmission. MeV vaccines are based on a live attenuated strain that indiscriminately enters cells expressing a ubiquitous protein, rather than sequentially entering target tissues through specific receptors. Ectopic replication of the vaccine strain alarms the immune system, which prevents overt disease. Because of the availability of a highly effective vaccine MeV is a candidate for eradication.

Influenza A virus (IAV) vaccines offer little protection from mismatched viruses with antigenically distant hemagglutinin (HA) glycoproteins. We sought to determine if a cationic lipid/DNA complex (CLDC) adjuvant could induce heterosubtypic protection if added to a whole inactivated IAV vaccine (WIV).Adult rhesus macaques (RMs) were vaccinated and at 2 weeks boosted with either an H1N1-WIV or an H3N2-WIV, with and without CLDC adjuvant. Four weeks postboost, animals were challenged with an H1N1 IAV matched to the H1N1-WIV vaccine.After challenge, viral RNA (vRNA) levels in the trachea of control RMs and RMs vaccinated with the unadjuvanted H1 or H3 WIV vaccines were similar. However, vRNA levels in the trachea of both the H1-WIV/CLDC- and the H3-WIV/CLDC-vaccinated RMs (P < 0.01 and P < 0.05, respectively) were significantly lower than in unvaccinated control RMs. Heterosubtypic protection in H3-WIV/CLDC RMs was associated with significantly higher levels of nucleoprotein (NP) and matrix-1-specific immunoglobulin G antibodies (P < 0.05) and NP-specific nonneutralizing antibody-dependent natural killer cell activation (P < 0.01) compared with unprotected H3-WIV RMs.Addition of the CLDC adjuvant to a simple WIV elicited immunity to conserved virus structural proteins in RMs that correlate with protection from uncontrolled virus replication after heterosubtypic influenza virus challenge.

Background:New methods to increase measles and rubella (MR) vaccination coverage are needed to achieve global and regional MR elimination goals. Methods:Here, we developed microneedle (MN) patches designed to administer MR vaccine by minimally trained personnel, leave no biohazardous sharps waste, remove the need for vaccine reconstitution, and provide thermostability outside the cold chain. This study evaluated the immunogenicity of MN patches delivering MR vaccine to infant rhesus macaques. Results:Protective titers of measles neutralizing antibodies (>120 mIU/mL) were detected in 100% of macaques in the MN group and 75% of macaques in the subcutaneous (SC) injection group. Rubella neutralizing antibody titers were >10 IU/mL for all groups. All macaques in the MN group were protected from challenge with wild-type measles virus, whereas 75% were protected in the SC group. However, vaccination by the MN or SC route was unable to generate protective immune responses to measles in infant macaques pretreated with measles immunoglobulin to simulate maternal antibody. Conclusions:These results show, for the first time, that MR vaccine delivered by MN patch generated protective titers of neutralizing antibodies to both measles and rubella in infant rhesus macaques and afforded complete protection from measles virus challenge.

Recombinant viral vectors are considered one of the major means for induction of strong immune responses against recombinant antigens by genetic immunization. Recently developed viral-vectored HIV vaccine candidates, despite achieving high levels transgene expression and inducing high magnitude immune responses to HIV, have faced limitations related to anti-vector immunity. In contrast, lentiviral vectors (LV) have been shown to be less sensitive to antivector neutralizing activity, while displaying desirable characteristics such as transduction of non-dividing cells, including antigenpresenting cells, and long-term transgene expression. We have developed a VSV-G-pseudotyped HIV-based lentiviral vaccine candidate, VRX1023, carrying unmodi ed HIV Gag, Pol and Rev genes derived from the HIV-1 NL4-3 strain and which expression is driven by the wild-type HIV LTR. In mice, this vector was shown to induce signi cant mucosal and systemic cellular and humoral responses against HIV after sub-cutaneous injection. In the present study, we investigated the ability of this vector to confer durable HIV-speci c immunogenicity for a time period of up to 1 year in the murine model. HIV-speci c immunity was tested in splenocytes from immunized animals, at various timepoints over a 12 month period postimmunization, by IFN-g Elispot. Gag and Pol-specific T-cell responses, assessed after stimulation with overlapping HIV-peptides, were sustained in the range of 800 spot forming units/million cells on average for over a year with no diminution over time. Rev-speci c immunity, although detected at lower levels, was also sustained, for up to 6 months post-immunization. Gag p24-speci c IgG levels could be detected at remarkably consistent levels from 2 weeks to 12 months post-vector administration. LV-speci c humoral immunity, speci cally targeted towards the VSV-G pseudotyping protein, was detected at high level throughout the course of the study, but was found to have minimal neutralizing activity. LV was successfully repeatedly administered without any marked increase of anti-vector neutralizing activity. In summary, VRX1023 represents a highly attractive HIV vaccine candidate that combines long-term immunogenicity with the ability to elicit weak host neutralizing activity despite repeated immunizations. VRX1023, in addition to competing favorably with existing vectors for anti-HIV immune responses, demonstrates unique features likely to address some of the pitfalls of current vector-based HIV vaccine strategies.

Measles is a highly contagious viral disease in NHP. The infection can range from asymptomatic to rapidly fatal, resulting in significant morbidity and mortality in captive populations. In addition to appropriate quarantine practices, restricted access, the immunization of all personnel in contact with NHP, and the wearing of protective clothing including face masks, measles immunization further reduces the infection risk. Commercially available measles vaccines are effective for use in NHP, but interruptions in their availability have prevented the implementation of ongoing, consistent vaccination programs. This need for a readily available vaccine led us to perform a broad, multicenter safety and immunogenicity study of another candidate vaccine, MVac (Serum Institute of India), a monovalent measles vaccine derived from live Edmonston-Zagreb strain virus that had been attenuated after 22 passages on human diploid cells.

Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis and conjunctivitis, and seroconversion before adolescence is common in humans. To gain some insight into how Ad5 infection affects the immune system of rhesus macaques (RM) 18 RM were infected with a host-range mutant Ad5 (Ad5hr) by 3 mucosal inoculations. There was a delay of 2 to 6 weeks after the first inoculation before plasmacytoid dendritic cell (pDC) frequency and function increased in peripheral blood. Primary Ad5hr infection suppressed IFN-γ mRNA expression, but the second Ad5hr exposure induced a rapid increase in IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC). Primary Ad5hr infection suppressed CCL20, TNF and IL-1 mRNA expression in PBMC, and subsequent virus exposures further dampened expression of these pro-inflammatory cytokines. Primary, but not secondary, Ad5hr inoculation increased the frequency of CXCR3+ CD4+ T cells in blood, while secondary, but not primary, Ad5hr infection transiently increased the frequencies of Ki67+, HLADR+ and CD95+/CCR5+ CD4+ T cells in blood. Ad5hr infection induced polyfunctional CD4 and CD8+ T cells specific for the Ad5 hexon protein in all of the animals. Thus, infection with Ad5hr induced a complex pattern of innate and adaptive immunity in RM that included transient systemic CD4+ T cell activation and suppressed innate immunity on re-exposure to the virus. The complex effects of adenovirus infection on the immune system may help to explain the unexpected results of testing Ad5 vector expressing HIV antigens in Ad5 seropositive people.

BACKGROUND The decreased immune response among elderly individuals results in reduced influenza vaccine efficacy. Strategies to improve vaccine efficacy in elderly individuals are needed. The goal of this study was to determine whether a cationic lipid/DNA complex (CLDC) can improve the efficacy of the trivalent inactivated influenza vaccine Fluzone in elderly nonhuman primates. METHODS Elderly (age, >18 years) rhesus macaques were vaccinated with Fluzone, with or without CLDC, and challenged with a human seasonal influenza virus isolate, A/Memphis/7/2001(H1N1). RESULTS We found that elderly macaques have significantly lower levels of circulating naive CD4(+) T cells, naive CD8(+) T cells, and B cells as compared to juvenile monkeys. Furthermore, on the day of challenge, recipients of Fluzone/CLDC had significantly higher plasma anti-influenza virus immunoglobulin G (P < .001) and immunoglobulin A (P < .001) titers than recipients of Fluzone alone. After virus challenge, only the Fluzone/CLDC-vaccinated animals had a significantly lower level of virus replication (P < .01) relative to the unvaccinated control animals. CONCLUSIONS These results demonstrate that CLDC can enhance the immunogenicity and efficacy of a licensed TIV in immunosenescent elderly monkeys.

Purpose of reviewThe development of a preventive HIV vaccine remains an unresolved challenge. Animal models that can predict the results of HIV vaccine efficacy trials and identify the immune mechanisms responsible for vaccine protection would be most useful for HIV vaccine development. The purpose of the current review is to critique recent developments in the use of animal models of HIV infection in preclinical studies of AIDS vaccines and to describe how the use of improved animal models can inform the development of an HIV vaccine.Recent findingsThe results of preclinical experiments with candidate HIV vaccines can vary with the SIV challenge virus used. It is now known that there is considerable variability in the neutralization sensitivity and that the level of viral sequence diversity within the challenge stocks varies. This has allowed more realistic preclinical vaccine studies with heterologous vaccine antigens and challenge viruses. Further, the dose of challenge virus and the route of virus challenge can modify the efficacy of candidate vaccines in preclinical studies.SummaryRecent experiments demonstrate that nonhuman primate models of AIDS can reproduce the complex biology of HIV transmission, recapitulate the results of HIV vaccine efficacy trials in humans and be used to identify correlates of protection.

PURPOSE OF REVIEW The development of a preventive HIV vaccine remains an unresolved challenge. Animal models that can predict the results of HIV vaccine efficacy trials and identify the immune mechanisms responsible for vaccine protection would be most useful for HIV vaccine development. The purpose of the current review is to critique recent developments in the use of animal models of HIV infection in preclinical studies of AIDS vaccines and to describe how the use of improved animal models can inform the development of an HIV vaccine. RECENT FINDINGS The results of preclinical experiments with candidate HIV vaccines can vary with the SIV challenge virus used. It is now known that there is considerable variability in the neutralization sensitivity and that the level of viral sequence diversity within the challenge stocks varies. This has allowed more realistic preclinical vaccine studies with heterologous vaccine antigens and challenge viruses. Further, the dose of challenge virus and the route of virus challenge can modify the efficacy of candidate vaccines in preclinical studies. SUMMARY Recent experiments demonstrate that nonhuman primate models of AIDS can reproduce the complex biology of HIV transmission, recapitulate the results of HIV vaccine efficacy trials in humans and be used to identify correlates of protection.

No information exists regarding immune responses to human immunodeficiency virus (HIV) infection in the foreskin or glans of the human penis, although this is a key tissue for HIV transmission. To address this gap, we characterized antiviral immune responses in foreskin of male rhesus macaques (RMs) inoculated with simian immunodeficiency virus (SIV) strain SIVmac251 by penile foreskin exposure. We found a complete population of immune cells in the foreskin and glans of normal RMs, although B cells were less common than CD4(+) and CD8(+) T cells. IgG-secreting cells were detected by enzyme-linked immunospot (ELISPOT) assay in cell suspensions made from the foreskin. In the foreskin and glans of SIV-infected RMs, although B cells were less common than CD4(+) and CD8(+) T cells, SIV-specific IgG antibody was present in foreskin secretions. In addition, cytokine-secreting SIV-specific CD8(+) T cells were readily found in cell suspensions made from the foreskin. Although potential HIV target cells were found in and under the epithelium covering all penile surfaces, the presence of antiviral effector B and T cells in the foreskin suggests that vaccines may be able to elicit immunity in this critical site to protect men from acquiring HIV.

ABSTRACT Immunization with attenuated lentiviruses is the only reliable method of protecting rhesus macaques (RM) from vaginal challenge with pathogenic simian immunodeficiency virus (SIV). CD8 + lymphocyte depletion prior to SIVmac239 vaginal challenge demonstrated that a modest, Gag-specific CD8 + T cell response induced by immunization with simian-human immunodeficiency virus 89.6 (SHIV89.6) protects RM. Although CD8 + T cells are required for protection, there is no anamnestic expansion of SIV-specific CD8 + T cells in any tissues except the vagina after challenge. Further, SHIV immunization increased the number of viral target cells in the vagina and cervix, suggesting that the ratio of target cells to antiviral CD8 + T cells was not a determinant of protection. We hypothesized that persistent replication of the attenuated vaccine virus modulates inflammatory responses and limits T cell activation and expansion by inducing immunoregulatory T cell populations. We found that attenuated SHIV infection decreased the number of circulating plasmacytoid dendritic cells, suppressed T cell activation, decreased mRNA levels of proinflammatory mediators, and increased mRNA levels of immunoregulatory molecules. Three days after SIV vaginal challenge, SHIV-immunized RM had significantly more T regulatory cells in the vagina than the unimmunized RM. By day 14 postchallenge, immune activation and inflammation were characteristic of unimmunized RM but were minimal in SHIV-immunized RM. Thus, a modest vaccine-induced CD8 + T cell response in the context of immunoregulatory suppression of T cell activation may protect against vaginal HIV transmission.

ABSTRACT The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8 + T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10 3 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.

This study sought to define the role of memory lymphocytes in the protection from homologous influenza A virus re-challenge in rhesus macaques. Depleting monoclonal antibodies (mAb) were administered to the animals prior to their second experimental inoculation with a human seasonal influenza A virus strain. Treatment with either anti-CD8α or anti-CD20 mAbs prior to re-challenge had minimal effect on influenza A virus replication. Thus, in non-human primates with pre-existing anti-influenza A antibodies, memory B cells and CD8α+ T cells do not contribute to the control of virus replication after re-challenge with a homologous strain of influenza A virus.

To determine the role of innate immune responses in controlling influenza A virus replication, rhesus macaques (RM) were administered pegylated IFN-alpha prior to virus challenge. Systemic and mucosal pegylated IFN-alpha administration induced expression of the interferon-stimulated genes (ISG) MxA and OAS in the airways. RM treated with IFN-alpha 24 hours prior to influenza virus challenge had significantly lower peak vRNA levels in the trachea compared to untreated animals. In addition to blunting viral replication, IFN-alpha treatment minimized the weight loss and spike in body temperature after influenza infection of RM. These results confirm the importance of IFN-alpha induced innate immune responses in the rapid control of influenza A virus replication in primates.

Venezuelan equine encephalitis virus replicon particles (VRP) without a transgene (null VRP) have been used to adjuvant effective humoral [1], cellular [2], and mucosal [3] immune responses in mice. To assess the adjuvant activity of null VRP in the context of a licensed inactivated influenza virus vaccine, rhesus monkeys were immunized with Fluzone® alone or Fluzone® mixed with null VRP and then challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). Compared to Fluzone® alone, Fluzone®+null VRP immunized animals had stronger influenza-specific CD4+ T cell responses (4.4 fold) with significantly higher levels of virus-specific IFN-γ (7.6 fold) and IL-2 (5.3 fold) producing CD4+ T cells. Fluzone®+null VRP immunized animals also had significantly higher plasma anti-influenza IgG (p<0.0001, 1.3 log) and IgA (p<0.05, 1.2 log) levels. In fact, the mean plasma anti-influenza IgG titers after one Fluzone®+null VRP immunization was 1.2 log greater (p<0.04) than after two immunizations with Fluzone® alone. After virus challenge, only Fluzone®+null VRP immunized monkeys had a significantly lower level of viral replication (p<0.001) relative to the unimmunized control animals. Although little anti-influenza antibody was detected in the respiratory secretions after immunization, strong anamnestic anti-influenza IgG and IgA responses were present in secretions of the Fluzone®+null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine.

Background In a previous study, progesterone treatment of female monkeys immunized with live, attenuated SHIV89.6 abrogated the generally consistent protection from vaginal simian immunodeficiency virus (SIV) challenge. The mechanisms responsible for the loss of protection remain to be defined. The objective of the present study was to determine whether Depo-Provera® administration alters protection from intravenous SIV challenge in SHIV-immunized female macaques. Methods and Findings Two groups of female macaques were immunized with attenuated SHIV89.6 and then challenged intravenously with SIVmac239. Four weeks before challenge, one animal group was treated with Depo-Provera®, a commonly used injectable contraceptive progestin. As expected, SHIV-immunized monkeys had significantly lower peak and set-point plasma viral RNA levels compared to naïve controls, but in contrast to previously published findings with vaginal SIV challenge, the Depo-Provera® SHIV-immunized animals controlled SIV replication to a similar, or even slightly greater, degree than did the untreated SHIV-immunized animals. Control of viral replication from week 4 to week 20 after challenge was more consistent in the progesterone-treated, SHIV-immunized animals than in untreated, SHIV-immunized animals. Although levels of interferon-γ production were similar, the SIV-specific CD8+ T cells of progesterone-treated animals expressed more functions than the anti-viral CD8+ T cells from untreated animals. Conclusions Depo-Provera® did not diminish the control of viral replication after intravenous SIV challenge in female macaques immunized with a live-attenuated lentivirus. This result contrasts with the previously reported effect of Depo-Provera® on protection from vaginal SIV challenge and strongly implies that the decreased protection from vaginal challenge is due to effects of progesterone on the genital tract rather than to systemic effects. Further, these results demonstrate that the effects of hormonal contraceptives on vaccine efficacy need to be considered in the context of testing and use of an AIDS vaccine.

The signaling lymphocytic activation molecule (SLAM; CD150) is the immune cell receptor for measles virus (MV). To assess the importance of the SLAM-MV interactions for virus spread and pathogenesis, we generated a wild-type IC-B MV selectively unable to recognize human SLAM (SLAM-blind). This virus differs from the fully virulent wild-type IC-B strain by a single arginine-to-alanine substitution at amino acid 533 of the attachment protein hemagglutinin and infects cells through SLAM about 40 times less efficiently than the isogenic wild-type strain. Ex vivo, this virus infects primary lymphocytes at low levels regardless of SLAM expression. When a group of six rhesus monkeys (Macaca mulatta) was inoculated intranasally with the SLAM-blind virus, no clinical symptoms were documented. Only one monkey had low-level viremia early after infection, whereas all the hosts in the control group had high viremia levels. Despite minimal, if any, viremia, all six hosts generated neutralizing antibody titers close to those of the control monkeys while MV-directed cellular immunity reached levels at least as high as in wild-type-infected monkeys. These findings prove formally that efficient SLAM recognition is necessary for MV virulence and pathogenesis. They also suggest that the selectively SLAM-blind wild-type MV can be developed into a vaccine vector.