Mutations in the F protein of the live-attenuated respiratory syncytial virus vaccine candidate ΔNS2/Δ1313/I1314L increase the stability of infectivity and content of prefusion F protein.

Respiratory syncytial virus (RSV) is the leading viral cause of bronchiolitis and pneumonia in infants and toddlers, but there currently is no licensed pediatric vaccine. A leading vaccine candidate that has been evaluated for intranasal immunization in a recently completed phase 1/2 clinical trial is an attenuated version of RSV strain A2 called RSV/ΔNS2/Δ1313/I1314L (hereafter called ΔNS2). ΔNS2 is attenuated by deletion of the interferon antagonist NS2 gene and introduction into the L polymerase protein gene of a codon deletion (Δ1313) that confers temperature-sensitivity and is stabilized by a missense mutation (I1314L). Previously, introduction of four amino acid changes derived from a second RSV strain "line 19" (I79M, K191R, T357K, N371Y) into the F protein of strain A2 increased the stability of infectivity and the proportion of F protein in the highly immunogenic pre-fusion (pre-F) conformation. In the present study, these four "line 19" assignments were introduced into the ΔNS2 candidate, creating ΔNS2-L19F-4M. During in vitro growth in Vero cells, ΔNS2-L19F-4M had growth kinetics and peak titer similar to the ΔNS2 parent. ΔNS2-L19F-4M exhibited an enhanced proportion of pre-F protein, with a ratio of pre-F/total F that was 4.5- to 5.0-fold higher than that of the ΔNS2 parent. The stability of infectivity during incubation at 4°C, 25°C, 32°C and 37°C was greater for ΔNS2-L19F-4M; for example, after 28 days at 32°C, its titer was 100-fold greater than ΔNS2. ΔNS2-L19F-4M exhibited similar levels of replication in human airway epithelial (HAE) cells as ΔNS2. The four "line 19" F mutations were genetically stable during 10 rounds of serial passage in Vero cells. In African green monkeys, ΔNS2-L19F-4M and ΔNS2 had similar growth kinetics, peak titer, and immunogenicity. These results suggest that ΔNS2-L19F-4M is an improved live attenuated vaccine candidate whose enhanced stability may simplify its manufacture, storage and distribution, which merits further evaluation in a clinical trial in humans.

Beta-containing bivalent SARS-CoV-2 protein vaccine elicits durable broad neutralization in macaques and protection in hamsters.

BACKGROUND: Since the beginning of the COVID-19 pandemic, several variants of concern (VOC) have emerged for which there is evidence of an increase in transmissibility, more severe disease, and/or reduced vaccine effectiveness. Effective COVID-19 vaccine strategies are required to achieve broad protective immunity against current and future VOC. METHODS: We conducted immunogenicity and challenge studies in macaques and hamsters using a bivalent recombinant vaccine formulation containing the SARS-CoV-2 prefusion-stabilized Spike trimers of the ancestral D614 and the variant Beta strains with AS03 adjuvant (CoV2 preS dTM-AS03) in a primary immunization setting. RESULTS: We show that a primary immunization with the bivalent CoV2 preS dTM-AS03 elicits broader and durable (1 year) neutralizing antibody responses against VOC including Omicron BA.1 and BA.4/5, and SARS-CoV-1 as compared to the ancestral D614 or Beta variant monovalent vaccines in naïve non-human primates. In addition, the bivalent formulation confers protection against viral challenge with SARS-CoV-2 prototype D614G strain as well as Alpha and Beta variant strains in hamsters. CONCLUSIONS: Our findings demonstrate the potential of a Beta-containing bivalent CoV2 preS dTM-AS03 formulation to provide broad and durable immunogenicity, as well as protection against VOC in naïve populations.

SARS-CoV-2 has changed over time, resulting in different forms of the virus called variants. These variants compromise the protection offered by the COVID-19 vaccines, which trigger an immune response against the viral Spike protein that allows the virus to attach and infect human cells, since their spike proteins are different. Here, we developed and tested a vaccine containing two different Spike proteins, one from the original Wuhan strain and another from the Beta variant. In macaques, the vaccine leads to the production of antibodies able to stop all variants tested from infecting human cells, including Omicron, with stable levels over one year. In hamsters, the vaccine protected against infection with the ancestral virus and the Alpha and Beta variants. Our findings have important implications for vaccine control of existing and future SARS-CoV-2 variants of concern.

Beta variant COVID-19 protein booster vaccine elicits durable cross-neutralization against SARS-CoV-2 variants in non-human primates.

The rapid spread of the SARS-CoV-2 Omicron subvariants, despite the implementation of booster vaccination, has raised questions about the durability of protection conferred by current vaccines. Vaccine boosters that can induce broader and more durable immune responses against SARS-CoV-2 are urgently needed. We recently reported that our Beta-containing protein-based SARS-CoV-2 spike booster vaccine candidates with AS03 adjuvant (CoV2 preS dTM-AS03) elicited robust cross-neutralizing antibody responses at early timepoints against SARS-CoV-2 variants of concern in macaques primed with mRNA or protein-based subunit vaccine candidates. Here we demonstrate that the monovalent Beta vaccine with AS03 adjuvant induces durable cross-neutralizing antibody responses against the prototype strain D614G as well as variants Delta (B.1.617.2), Omicron (BA.1 and BA.4/5) and SARS-CoV-1, that are still detectable in all macaques 6 months post-booster. We also describe the induction of consistent and robust memory B cell responses, independent of the levels measured post-primary immunization. These data suggest that a booster dose with a monovalent Beta CoV2 preS dTM-AS03 vaccine can induce robust and durable cross-neutralizing responses against a broad spectrum of variants.

Immunogenicity and protective efficacy of RSV G central conserved domain vaccine with a prefusion nanoparticle.

Respiratory syncytial virus (RSV) G glycoprotein has recently reemerged as a vaccine antigen due to its ability to elicit potent neutralizing antibodies and ameliorate disease in animal models. Here we designed three constructs to display the G central conserved domain (Gcc) focused on inducing broad and potent neutralizing antibodies. One construct displaying Gcc from both RSV subgroups trimerized via a C-terminal foldon (Gcc-Foldon) was highly immunogenic in mice and in MIMIC, a pre-immune human in vitro model. To explore an optimal RSV vaccine, we combined the Gcc-Foldon antigen with a stabilized pre-fusion-F nanoparticle (pre-F-NP) as a bivalent vaccine and detected no antigenic interference between the two antigens in the MIMIC model. In RSV-primed macaques, the bivalent vaccine elicited potent humoral responses. Furthermore, both Gcc-Foldon and the bivalent vaccine conferred effective protection against RSV challenge in mice. This two-component vaccine could potentially provide effective protection against RSV infection in humans and warrants further clinical evaluation.

Protein-based SARS-CoV-2 spike vaccine booster increases cross-neutralization against SARS-CoV-2 variants of concern in non-human primates.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that partly evade neutralizing antibodies raises concerns of reduced vaccine effectiveness and increased infection. We previously demonstrated that the SARS-CoV-2 spike protein vaccine adjuvanted with AS03 (CoV2 preS dTM-AS03) elicits robust neutralizing antibody responses in naïve subjects. Here we show that, in macaques primed with mRNA or protein-based subunit vaccine candidates, one booster dose of CoV2 preS dTM-AS03 (monovalent D614 or B.1.351, or bivalent D614 + B.1.351 formulations), significantly boosts the pre-existing neutralizing antibodies against the parental strain from 177- to 370-fold. Importantly, the booster dose elicits high and persistent cross-neutralizing antibodies covering five former or current SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta and Omicron) and, unexpectedly, SARS-CoV-1. Interestingly, we show that the booster specifically increases the functional antibody responses as compared to the receptor binding domain (RBD)-specific responses. Our findings show that these vaccine candidates, when used as a booster, have the potential to offer cross-protection against a broad spectrum of variants. This has important implications for vaccine control of SARS-CoV-2 variants of concern and informs on the benefit of a booster with the vaccine candidates currently under evaluation in clinical trials.

Pan-SARS neutralizing responses after third boost vaccination in non-human primate immunogenicity model.

The emergence of SARS-CoV-2 variants, especially Beta and Delta, has raised concerns about the reduced protection from previous infection or vaccination based on the original Wuhan-Hu-1 (D614) virus. To identify promising regimens for inducing neutralizing titers towards new variants, we evaluated monovalent and bivalent mRNA vaccines either as primary vaccination or as a booster in nonhuman primates (NHPs). Two mRNA vaccines, D614-based MRT5500 and Beta-based MRT5500ß, tested in sequential regimens or as a bivalent combination in naïve NHPs produced modest neutralizing titers to heterologous variants. However, when mRNA vaccines were administered as a booster to pre-immune NHPs, we observed a robust increase in neutralizing titers with expanded breadth towards all tested variants, and notably SARS-CoV-1. The breadth of the neutralizing response was independent of vaccine sequence or modality, as we further showed either MRT5500 or recombinant subunit Spike protein (with adjuvant) can serve as boosters to induce broadly neutralizing antibodies in the NHPs primed with MRT5500. The data support the notion that a third vaccination is key to boosting existing titers and improving the breadth of antibodies to address variants of concern, including those with an E484K mutation in the Receptor Binding Domain (RBD) (Beta, Gamma).

Immunogenicity and efficacy of mRNA COVID-19 vaccine MRT5500 in preclinical animal models.

Emergency use authorization of COVID vaccines has brought hope to mitigate pandemic of coronavirus disease 2019 (COVID-19). However, there remains a need for additional effective vaccines to meet the global demand and address the potential new viral variants. mRNA technologies offer an expeditious path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on the spike (S) glycoprotein of SARS-CoV-2. Several mRNA constructs of S-protein, including wild type, a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), as well as others, were tested in animal models for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques, and for efficacy in a Syrian golden hamster model. The selected 2P/GSAS vaccine formulation, designated MRT5500, elicited potent nAbs as measured in neutralization assays in all three preclinical models and more importantly, protected against SARS-CoV-2-induced weight loss and lung pathology in hamsters. In addition, MRT5500 elicited TH1-biased responses in both mouse and non-human primate (NHP), thus alleviating a hypothetical concern of potential vaccine-associated enhanced respiratory diseases known associated with TH2-biased responses. These data position MRT5500 as a viable vaccine candidate for entering clinical development.

Evaluation of the respiratory syncytial virus G-directed neutralizing antibody response in the human airway epithelial cell model.

Human respiratory syncytial virus (RSV) is a major cause of serious respiratory tract infections in infants and the elderly. Recently it was shown that the RSV G glycoprotein mediates attachment to cells using CX3CR1 as a receptor, and that G-specific neutralizing antibodies can be detected using human airway epithelial (HAE) cell cultures. To investigate the contributions of G-specific antibodies to RSV neutralization, we performed HAE neutralization assays on sera from RSV G-immunized mice or RSV-infected infants. We confirmed that G-specific neutralization using serum from mice or humans could only be detected on HAE cultures. We also found that RSV G-specific antibodies in infants were either subgroup specific or cross-neutralizing. Altogether, our results suggest that G is an important target for generating neutralizing antibodies and would be beneficial to include in an RSV vaccine. Further, inclusion of G antigens from both RSV subgroups may enhance the vaccine cross protection potency.

A respiratory syncytial virus (RSV) F protein nanoparticle vaccine focuses antibody responses to a conserved neutralization domain.

A stabilized form of the respiratory syncytial virus (RSV) fusion (F) protein has been explored as a vaccine to prevent viral infection because it presents several potent neutralizing epitopes. Here, we used a structure-based rational design to optimize antigen presentation and focus antibody (Ab) responses to key epitopes on the pre-fusion (pre-F) protein. This protein was fused to ferritin nanoparticles (pre-F-NP) and modified with glycans to mask nonneutralizing or poorly neutralizing epitopes to further focus the Ab response. The multimeric pre-F-NP elicited durable pre-F-specific Abs in nonhuman primates (NHPs) after >150 days and elicited potent neutralizing Ab (NAb) responses in mice and NHPs in vivo, as well as in human cells evaluated in the in vitro MIMIC system. This optimized pre-F-NP stimulated a more potent Ab response than a representative pre-F trimer, DS-Cav1. Collectively, this pre-F vaccine increased the generation of NAbs targeting the desired pre-F conformation, an attribute that facilitates the development of an effective RSV vaccine.

Effect of genetic background and delivery route on the preclinical properties of a live attenuated RSV vaccine.

A safe and effective vaccine against RSV remains an important unmet public health need. Intranasally (IN) delivered live-attenuated vaccines represent the most extensively studied approach for immunization of RSV-naïve infants and children, however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we report pre-clinical immunogenicity and efficacy data utilizing a live-attenuated vaccine candidate, RGΔM2-2, which was obtained by deleting the M2-2 open reading frame from the genome of the MSA1 clinical isolate. Intramuscular (IM) administration of RGΔM2-2 in cotton rats induced immunity and protective efficacy that was comparable to that induced by intranasal (IN) immunization. In contrast, the protective efficacy of RGΔM2-2 delivered by the IM route to African green monkeys was substantially reduced as compared to the efficacy following IN administration, despite comparable levels of serum neutralizing antibodies. This result suggests that mucosal immunity may play an important role in RSV protection. The RGΔM2-2 vaccine also demonstrated different attenuation profiles when tested in cotton rats, non-human primates, and a human airway epithelial (HAE) cell model. The data suggest RGΔM2-2 is less attenuated than a similarly designed vaccine candidate constructed on the A2 genetic background. These findings have important implications with regard to both the design and the preclinical safety testing of live-attenuated vaccines.

Core bead chromatography for preparation of highly pure, infectious respiratory syncytial virus in the negative purification mode.

Respiratory syncytial virus (RSV) is an important human pathogen, and is the most frequent viral cause of severe respiratory disease in infants. In addition, it is increasingly being recognized as an important cause of respiratory disease in the elderly and immunocompromised. Although a passive prophylactic treatment does exist for high-risk neonates and children, the overall disease burden warrants the development of a safe and effective prophylactic vaccine for use in otherwise healthy newborns and children. RSV is known to be an extremely labile virus, prone to aggregation and loss of infectious titer during virus handling and preparation procedures. To date infective RSV virions have been prepared by methods which are not readily scalable, such as density gradient ultracentrifugation. In this study we describe a scalable, chromatography-based purification procedure for preparation of highly pure, infectious RSV. The purification scheme is based on core bead technology and hollow fiber tangential flow filtration (TFF) and results in a ∼60% recovery of infectious virus titer. This method can be used to prepare highly purified wild type or live-attenuated vaccine strain viruses with titers as high as 1×10(8) plaque forming units per mL. A live-attenuated RSV vaccine prepared by this method was found to be immunogenic and protective in vivo, and its purity was 50-200-fold greater with respect to host cell dsDNA and Vero host cell proteins, than the raw feed stream. The results presented here can be considered a starting point for downstream process development of a live-attenuated vaccine approach for prevention of disease by RSV.

CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner.

Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein's CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.

Pilot study on the immunogenicity of paired Env immunogens from mother-to-child transmitted HIV-1 isolates.

Recent studies have reported that founder viruses play unique roles in establishing HIV-1 infection. Understanding the biological and immunological features of envelope glycoproteins (Env) from such viruses may facilitate the development of effective vaccines against HIV-1. In this report, we evaluated the immunogenicity of gp120 immunogens from two pairs of clade B and two pairs of clade C mother-to-child transmitted (MTCT) HIV-1 variants that had various levels of sensitivity to broadly neutralizing monoclonal antibodies. Individual gp120 DNA and protein vaccines were produced from each of the eight MTCT Env antigens included in the current study. Rabbits were immunized with these gp120 immunogens by the DNA prime-protein boost approach. High level Env-specific antibody responses were elicited by all MTCT gp120 immunogens. However, their abilities to elicit neutralizing antibody (NAb) responses differed and those from relatively neutralization-resistant variants tended to be more effective in eliciting broader NAb. Results of this pilot study indicated that not all MTCT Env proteins have the same potential to elicit NAb. Understanding the mechanism(s) behind such variation may provide useful information in formulating the next generation of HIV vaccines.

Genotypic and functional properties of early infant HIV-1 envelopes.

BACKGROUND: Understanding the properties of HIV-1 variants that are transmitted from women to their infants is crucial to improving strategies to prevent transmission. In this study, 162 full-length envelope (env) clones were generated from plasma RNA obtained from 5 HIV-1 Clade B infected mother-infant pairs. Following extensive genotypic and phylogenetic analyses, 35 representative clones were selected for functional studies. RESULTS: Infant quasispecies were highly homogeneous and generally represented minor maternal variants, consistent with transmission across a selective bottleneck. Infant clones did not differ from the maternal in env length, or glycosylation. All infant variants utilized the CCR5 co-receptor, but were not macrophage tropic. Relatively high levels (IC50 ≥ 100 µg/ml) of autologous maternal plasma IgG were required to neutralize maternal and infant viruses; however, all infant viruses were neutralized by pooled sera from HIV-1 infected individuals, implying that they were not inherently neutralization-resistant. All infant viruses were sensitive to the HIV-1 entry inhibitors Enfuvirtide and soluble CD4; none were resistant to Maraviroc. Sensitivity to human monoclonal antibodies 4E10, 2F5, b12 and 2G12 varied. CONCLUSIONS: This study provides extensive characterization of the genotypic and functional properties of HIV-1 env shortly after transmission. We present the first detailed comparisons of the macrophage tropism of infant and maternal env variants and their sensitivity to Maraviroc, the only CCR5 antagonist approved for therapeutic use. These findings may have implications for improving approaches to prevent mother-to-child HIV-1 transmission.

Immunogenicity and protection efficacy of subunit-based smallpox vaccines using variola major antigens.

The viral strain responsible for smallpox infection is variola major (VARV). As a result of the successful eradication of smallpox with the vaccinia virus (VACV), the general population is no longer required to receive a smallpox vaccine, and will have no protection against smallpox. This lack of immunity is a concern due to the potential for use of smallpox as a biological weapon. Considerable progress has been made in the development of subunit-based smallpox vaccines resulting from the identification of VACV protective antigens. It also offers the possibility of using antigens from VARV to formulate the next generation subunit-based smallpox vaccines. Here, we show that codon-optimized DNA vaccines expressing three VARV antigens (A30, B7 and F8) and their recombinant protein counterparts elicited high-titer, cross-reactive, VACV neutralizing antibody responses in mice. Vaccinated mice were protected from intraperitoneal and intranasal challenges with VACV. These results suggest the feasibility of a subunit smallpox vaccine based on VARV antigen sequences to induce immunity against poxvirus infection.

Immunogenicity of recombinant fiber-chimeric adenovirus serotype 35 vector-based vaccines in mice and rhesus monkeys.

Preexisting immunity to adenovirus serotype 5 (Ad5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. A potential solution to this problem is to utilize rAd vectors derived from rare Ad serotypes, such as Ad35. However, rAd35 vectors have appeared less immunogenic than rAd5 vectors in preclinical studies to date. In this study, we explore the hypothesis that the differences in immunogenicity between rAd5 and rAd35 vectors may be due in part to differences between the fiber proteins of these viruses. We constructed capsid chimeric rAd35 vectors containing the Ad5 fiber knob (rAd35k5) and compared the immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing simian immunodeficiency virus Gag and HIV-1 Env in mice and rhesus monkeys. In vitro studies demonstrated that rAd35k5 vectors utilized the Ad5 receptor CAR rather than the Ad35 receptor CD46. In vivo studies showed that rAd35k5 vectors were more immunogenic than rAd35 vectors in both mice and rhesus monkeys. These data suggest that the Ad5 fiber knob contributes substantially to the immunogenicity of rAd vectors. Moreover, these studies demonstrate that capsid chimeric rAd vectors can be constructed to combine beneficial immunologic and serologic properties of different Ad serotypes.

A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates.

Plasmid DNA vaccines elicit potent and protective immune responses in numerous small-animal models of infectious diseases. However, their immunogenicity in primates appears less potent. Here we investigate a novel approach that optimizes regulatory elements in the plasmid backbone to improve the immunogenicity of DNA vaccines. Among various regions analyzed, we found that the addition of a regulatory sequence from the R region of the long terminal repeat from human T-cell leukemia virus type 1 (HTLV-1) to the cytomegalovirus (CMV) enhancer/promoter increased transgene expression 5- to 10-fold and improved cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens. In cynomolgus monkeys, DNA vaccines containing the CMV enhancer/promoter with the HTLV-1 R region (CMV/R) induced markedly higher cellular immune responses to HIV-1 Env from clades A, B, and C and to HIV-1 Gag-Pol-Nef compared with the parental DNA vaccines. These data demonstrate that optimization of specific regulatory elements can substantially improve the immunogenicity of DNA vaccines encoding multiple antigens in small animals and in nonhuman primates. This strategy could therefore be explored as a potential method to enhance DNA vaccine immunogenicity in humans.

Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus hexon protein.

The utility of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens will likely be limited by the high prevalence of pre-existing Ad5-specific neutralizing Abs (NAbs) in human populations. However, the immunodominant targets of Ad5-specific NAbs in humans remain poorly characterized. In this study, we assess the titers and primary determinants of Ad5-specific NAbs in individuals from both the United States and the developing world. Importantly, median Ad5-specific NAb titers were >10-fold higher in sub-Saharan Africa compared with the United States. Moreover, hexon-specific NAb titers were 4- to 10-fold higher than fiber-specific NAb titers in these cohorts by virus neutralization assays using capsid chimeric viruses. We next performed adoptive transfer studies in mice to evaluate the functional capacity of hexon- and fiber-specific NAbs to suppress the immunogenicity of a prototype rAd5-Env vaccine. Hexon-specific NAbs were remarkably efficient at suppressing Env-specific immune responses elicited by the rAd5 vaccine. In contrast, fiber-specific NAbs exerted only minimal suppressive effects on rAd5 vaccine immunogenicity. These data demonstrate that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein in both humans and mice. These studies suggest a potential strategy for engineering novel Ad5 vectors to evade dominant Ad5-specific NAbs.

Dynamic immune responses maintain cytotoxic T lymphocyte epitope mutations in transmitted simian immunodeficiency virus variants.

Viral escape from cytotoxic T lymphocytes (CTLs) can undermine immune control of human immunodeficiency virus 1. It is therefore important to assess the stability of viral mutations in CTL epitopes after transmission to naive hosts. Here we demonstrate the persistence of mutations in a dominant CTL epitope after transmission of simian immunodeficiency virus variants to major histocompatibility complex-matched rhesus monkeys. Transient reversions to wild-type sequences occurred and elicited CTLs specific for the wild-type epitope, resulting in immunological pressure that rapidly reselected the mutant viruses. These data suggest that mutations in dominant human immunodeficiency virus 1 CTL epitopes may accumulate in human populations with limited major histocompatibility complex heterogeneity by a mechanism involving dynamic CTL control of transiently reverted wild-type virus.

Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines.

DCs are critical for priming adaptive immune responses to foreign antigens. However, the utility of harnessing these cells in vivo to optimize the immunogenicity of vaccines has not been fully explored. Here we investigate a novel vaccine approach that involves delivering synergistic signals that both recruit and expand DC populations at the site of antigen production. Intramuscular injection of an unadjuvanted HIV-1 envelope (env) DNA vaccine recruited few DCs to the injection site and elicited low-frequency, env-specific immune responses in mice. Coadministration of plasmids encoding the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) and the DC-specific growth factor fms-like tyrosine kinase 3 ligand with the DNA vaccine resulted in the recruitment, expansion, and activation of large numbers of DCs at the site of inoculation. Consistent with these findings, coadministration of these plasmid cytokines also markedly augmented DNA vaccine---elicited cellular and humoral immune responses and increased protective efficacy against challenge with recombinant vaccinia virus. These data suggest that the availability of mature DCs at the site of inoculation is a critical rate-limiting factor for DNA vaccine immunogenicity. Synergistic recruitment and expansion of DCs in vivo may prove a practical strategy for overcoming this limitation and potentiating immune responses to vaccines as well as other immunotherapeutic strategies.