Charge neutralized poly(ß-amino ester) polyplex nanoparticles for delivery of self-amplifying RNA.

Therapeutic self-amplifying RNA (saRNA) is a promising approach for disease treatment, as it can be administered in lower doses than messenger RNA (mRNA) to achieve comparable protein production levels. However, saRNA requires an appropriate delivery vehicle to protect it during transit and facilitate its transfection. A widely-adopted approach has been to use polycations to condense these large anionic macromolecules into polyplex nanoparticles, however their high charge density often elicits cytotoxic effects. In this study we postulated that we could improve the potency and tolerability of such delivery vehicles by co-formulating poly(ß-amino ester)s saRNA polyplexes with a non-toxic anionic polymer, γ-polyglutamic acid (γ-PGA) to neutralize partially this positive charge. Accordingly, we prepared a poly(ß-amino ester) from 1,6-hexanedioldiacrylate (HDDA) and 4-aminobutanol (ABOL) and initially evaluated the physicochemical properties of the binary polyplexes (i.e. formed from polymer and saRNA only). Optimised binary polyplex formulations were then taken forward for preparation of ternary complexes containing pHDDA-ABOL, saRNA and γ-PGA. Our findings demonstrate that γ-PGA integration into polyplexes significantly enhanced transfection efficacy in HEK293T and A431 cells without affecting polyplex size. Notably, γ-PGA incorporation leads to a pronounced reduction in zeta potential, which reduced the toxicity of the ternary complexes in moDC, NIH3T3, and A431 cells. Furthermore, the presence of γ-PGA contributed to colloidal stability, reducing aggregation of the ternary complexes, as evidenced by insignificant changes in polydispersity index (PDI) after freeze-thaw cycles. Overall, these results suggest that incorporating the appropriate ratio of a polyanion such as γ-PGA with polycations in RNA delivery formulations is a promising way to improve the in vitro delivery of saRNA.

Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

Immunogenicity of stabilized HIV-1 Env trimers delivered by self-amplifying mRNA.

Self-amplifying mRNA (saRNA) represents a promising platform for nucleic acid delivery of vaccine immunogens. Unlike plasmid DNA, saRNA does not require entry into the nucleus of target cells for expression, having the capacity to drive higher protein expression compared to mRNA as it replicates within the cytoplasm. In this study, we examined the potential of stabilized native-like HIV-1 Envelope glycoprotein (Env) trimers to elicit immune responses when delivered by saRNA polyplexes (PLXs), assembled with linear polyethylenimine. We showed that Venezuelan equine encephalitis virus (VEEV) saRNA induces a stronger humoral immune response to the encoded transgene compared to Semliki Forest virus saRNA. Moreover, we characterized the immunogenicity of the soluble and membrane-bound ConSOSL.UFO Env design in mice and showed a faster humoral kinetic and an immunoglobulin G (IgG)2a skew using a membrane-bound design. The immune response generated by PLX VEEV saRNA encoding the membrane-bound Env was then evaluated in larger animal models including macaques, in which low doses induced high IgG responses. Our data demonstrated that the VEEV saRNA PLX nanoparticle formulation represents a suitable platform for the delivery of stabilized HIV-1 Env and has the potential to be used in a variety of vaccine regimens.

Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines.

Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.

Heterologous vaccination regimens with self-amplifying RNA and adenoviral COVID vaccines induce robust immune responses in mice.

Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two-dose heterologous vaccination regimens than single-dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+ CD4 T cells, which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.

Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA.

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus (RABV)-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC50) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.

Neutrophils Enable Local and Non-Invasive Liposome Delivery to Inflamed Skeletal Muscle and Ischemic Heart.

Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease, and cancer. Improving localized delivery of immunosuppressive drugs to inflamed tissue in a non-invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Here, a neutrophil-mediated delivery system able to transport drug-loaded nanocarriers to inflamed tissue by exploiting the inherent ability of neutrophils to migrate to inflammatory tissue is reported. This hybrid system (neutrophils loaded with liposomes ex vivo) efficiently migrates in vitro following an inflammatory chemokine gradient. Furthermore, the triggered release of loaded liposomes and reuptake by target macrophages is studied. The migratory behavior of liposome-loaded neutrophils is confirmed in vivo by demonstrating the delivery of drug-loaded liposomes to an inflamed skeletal muscle in mice. A single low-dose injection of the hybrid system locally reduces inflammatory cytokine levels. Biodistribution of liposome-loaded neutrophils in a human-disease-relevant myocardial ischemia reperfusion injury mouse model after i.v. injection confirms the ability of injected neutrophils to carry loaded liposomes to inflammation sites. This strategy shows the potential of nanocarrier-loaded neutrophils as a universal platform to deliver anti-inflammatory drugs to promote tissue regeneration in inflammatory diseases.

The In Vitro, Ex Vivo, and In Vivo Effect of Polymer Hydrophobicity on Charge-Reversible Vectors for Self-Amplifying RNA.

RNA technology has the potential to revolutionize vaccination. However, the lack of clear structure-property relationships in relevant biological models mean there is no clear consensus on the chemical motifs necessary to improve RNA delivery. In this work, we describe the synthesis of a series of copolymers based on the self-hydrolyzing charge-reversible polycation poly(dimethylaminoethyl acrylate) (pDMAEA), varying the lipophilicity of the additional co-monomers. All copolymers formed stable polyplexes, showing efficient complexation with model nucleic acids from nitrogen/phosphate (N/P) ratios of N/P = 5, with more hydrophobic complexes exhibiting slower charge reversal and disassembly compared to hydrophilic analogues. The more hydrophobic copolymers outperformed hydrophilic versions, homopolymer controls and the reference standard polymer (polyethylenimine), in transfection assays on 2D cell monolayers, albeit with significantly higher toxicities. Similarly, hydrophobic derivatives displayed up to a 4-fold higher efficacy in terms of the numbers of cells expressing green fluorescent protein (GFP+) cells in ex vivo human skin (10%) compared to free RNA (2%), attributed to transfection enrichment in epithelial cells. In contrast, in a mouse model, we observed the reverse trend in terms of RNA transfection, with no observable protein production in more hydrophobic analogues, whereas hydrophilic copolymers induced the highest transfection in vivo. Overall, our results suggest an important relationship between the vector lipophilicity and RNA transfection in vaccine settings, with polymer biocompatibility potentially a key parameter in effective in vivo protein production.

Mannosylated Poly(ethylene imine) Copolymers Enhance saRNA Uptake and Expression in Human Skin Explants.

Messenger RNA (mRNA) is a promising platform for both vaccines and therapeutics, and self-amplifying RNA (saRNA) is particularly advantageous, as it enables higher protein expression and dose minimization. Here, we present a delivery platform for targeted delivery of saRNA using mannosylated poly(ethylene imine) (PEI) enabled by the host-guest interaction between cyclodextrin and adamantane. We show that the host-guest complexation does not interfere with the electrostatic interaction with saRNA and observed that increasing the degree of mannosylation inhibited transfection efficiency in vitro, but enhanced the number of cells expressing GFP by 8-fold in human skin explants. Besides, increasing the ratio of glycopolymer to saRNA also enhanced the percentage of transfected cells ex vivo. We identified that these mannosylated PEIs specifically increased protein expression in the epithelial cells resident in human skin in a mannose-dependent manner. This platform is promising for further study of glycosylation of PEI and targeted saRNA delivery.

Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA.

Self-amplifying RNA (saRNA) is a promising biotherapeutic tool that has been used as a vaccine against both infectious diseases and cancer. saRNA has been shown to induce protein expression for up to 60 days and elicit immune responses with lower dosing than messenger RNA (mRNA). Because saRNA is a large (~9500 nt), negatively charged molecule, it requires a delivery vehicle for efficient cellular uptake and degradation protection. Lipid nanoparticles (LNPs) have been widely used for RNA formulations, where the prevailing paradigm is to encapsulate RNA within the particle, including the first FDA-approved small-interfering siRNA therapy. Here, we compared LNP formulations with cationic and ionizable lipids with saRNA either on the interior or exterior of the particle. We show that LNPs formulated with cationic lipids protect saRNA from RNAse degradation, even when it is adsorbed to the surface. Furthermore, cationic LNPs deliver saRNA equivalently to particles formulated with saRNA encapsulated in an ionizable lipid particle, both in vitro and in vivo. Finally, we show that cationic and ionizable LNP formulations induce equivalent antibodies against HIV-1 Env gp140 as a model antigen. These studies establish formulating saRNA on the surface of cationic LNPs as an alternative to the paradigm of encapsulating RNA.

Combined Influence of B-Cell Receptor Rearrangement and Somatic Hypermutation on B-Cell Class-Switch Fate in Health and in Chronic Lymphocytic Leukemia.

A diverse B-cell receptor (BCR) repertoire is required to bind a wide range of antigens. BCRs are generated through genetic recombination and can be diversified through somatic hypermutation (SHM) or class-switch recombination (CSR). Patterns of repertoire diversity can vary substantially between different health conditions. We use isotype-resolved BCR sequencing to compare B-cell evolution and class-switch fate in healthy individuals and in patients with chronic lymphocytic leukemia (CLL). We show that the patterns of SHM and CSR in B-cells from healthy individuals are distinct from CLL. We identify distinct properties of clonal expansion that lead to the generation of antibodies of different classes in healthy, malignant, and non-malignant CLL BCR repertoires. We further demonstrate that BCR diversity is affected by relationships between antibody variable and constant regions leading to isotype-specific signatures of variable gene usage. This study provides powerful insights into the mechanisms underlying the evolution of the adaptive immune responses in health and their aberration during disease.

One Size Does Not Fit All: The Effect of Chain Length and Charge Density of Poly(ethylene imine) Based Copolymers on Delivery of pDNA, mRNA, and RepRNA Polyplexes.

Nucleic acid delivery systems are commonly translated between different modalities, such as DNA and RNA of varying length and structure, despite physical differences in these molecules that yield disparate delivery efficiency with the same system. Here, we synthesized a library of poly(2-ethyl-2-oxazoline)/poly(ethylene imine) copolymers with varying molar mass and charge densities in order to probe how pDNA, mRNA, and RepRNA polyplex characteristics affect transfection efficiency. The library was utilized in a full factorial design of experiment (DoE) screening, with outputs of luciferase expression, particle size, surface charge, and particle concentration. The optimal copolymer molar mass and charge density was found as 83 kDa/100%, 72 kDa/100%, and 45 kDa/80% for pDNA, RepRNA, and mRNA, respectively. While 10 of the synthesized copolymers enhanced the transfection efficiency of pDNA and mRNA, only 2 copolymers enhanced RepRNA transfection efficiency, indicating a narrow and more stringent design space for RepRNA. These findings suggest that there is not a "one size fits all" polymer for different nucleic acid species.

A Multi-Component Prime-Boost Vaccination Regimen with a Consensus MOMP Antigen Enhances Chlamydia trachomatis Clearance.

BACKGROUND: A vaccine for Chlamydia trachomatis is of urgent medical need. We explored bioinformatic approaches to generate an immunogen against C. trachomatis that would induce cross-serovar T-cell responses as (i) CD4(+) T cells have been shown in animal models and human studies to be important in chlamydial protection and (ii) antibody responses may be restrictive and serovar specific. METHODS: A consensus antigen based on over 1,500 major outer membrane protein (MOMP) sequences provided high epitope coverage against the most prevalent C. trachomatis strains in silico. Having designed the T-cell immunogen, we assessed it for immunogenicity in prime-boost regimens. This consensus MOMP transgene was delivered using plasmid DNA, Human Adenovirus 5 (HuAd5) or modified vaccinia Ankara (MVA) vectors with or without MF59(®) adjuvanted recombinant MOMP protein. RESULTS: Different regimens induced distinct immune profiles. The DNA-HuAd5-MVA-Protein vaccine regimen induced a cellular response with a Th1-biased serum antibody response, alongside high serum and vaginal MOMP-specific antibodies. This regimen significantly enhanced clearance against intravaginal C. trachomatis serovar D infection in both BALB/c and B6C3F1 mouse strains. This enhanced clearance was shown to be CD4(+) T-cell dependent. Future studies will need to confirm the specificity and precise mechanisms of protection. CONCLUSION: A C. trachomatis vaccine needs to induce a robust cellular response with broad cross-serovar coverage and a heterologous prime-boost regimen may be an approach to achieve this.

Intramuscular Immunisation with Chlamydial Proteins Induces Chlamydia trachomatis Specific Ocular Antibodies.

BACKGROUND: Ocular infection with Chlamydia trachomatis can cause trachoma, which is the leading cause of blindness due to infection worldwide. Despite the large-scale implementation of trachoma control programmes in the majority of countries where trachoma is endemic, there remains a need for a vaccine. Since C. trachomatis infects the conjunctival epithelium and stimulates an immune response in the associated lymphoid tissue, vaccine regimens that enhance local antibody responses could be advantageous. In experimental infections of non-human primates (NHPs), antibody specificity to C. trachomatis antigens was found to change over the course of ocular infection. The appearance of major outer membrane protein (MOMP) specific antibodies correlated with a reduction in ocular chlamydial burden, while subsequent generation of antibodies specific for PmpD and Pgp3 correlated with C. trachomatis eradication. METHODS: We used a range of heterologous prime-boost vaccinations with DNA, Adenovirus, modified vaccinia Ankara (MVA) and protein vaccines based on the major outer membrane protein (MOMP) as an antigen, and investigated the effect of vaccine route, antigen and regimen on the induction of anti-chlamydial antibodies detectable in the ocular lavage fluid of mice. RESULTS: Three intramuscular vaccinations with recombinant protein adjuvanted with MF59 induced significantly greater levels of anti-MOMP ocular antibodies than the other regimens tested. Intranasal delivery of vaccines induced less IgG antibody in the eye than intramuscular delivery. The inclusion of the antigens PmpD and Pgp3, singly or in combination, induced ocular antigen-specific IgG antibodies, although the anti-PmpD antibody response was consistently lower and attenuated by combination with other antigens. CONCLUSIONS: If translatable to NHPs and/or humans, this investigation of the murine C. trachomatis specific ocular antibody response following vaccination provides a potential mouse model for the rapid and high throughput evaluation of future trachoma vaccines.

Mucosal application of gp140 encoding DNA polyplexes to different tissues results in altered immunological outcomes in mice.

Increasing evidence suggests that mucosally targeted vaccines will enhance local humoral and cellular responses whilst still eliciting systemic immunity. We therefore investigated the capacity of nasal, sublingual or vaginal delivery of DNA-PEI polyplexes to prime immune responses prior to mucosal protein boost vaccination. Using a plasmid expressing the model antigen HIV CN54gp140 we show that each of these mucosal surfaces were permissive for DNA priming and production of antigen-specific antibody responses. The elicitation of systemic immune responses using nasally delivered polyplexed DNA followed by recombinant protein boost vaccination was equivalent to a systemic prime-boost regimen, but the mucosally applied modality had the advantage in that significant levels of antigen-specific IgA were detected in vaginal mucosal secretions. Moreover, mucosal vaccination elicited both local and systemic antigen-specific IgG(+) and IgA(+) antibody secreting cells. Finally, using an Influenza challenge model we found that a nasal or sublingual, but not vaginal, DNA prime/protein boost regimen protected against infectious challenge. These data demonstrate that mucosally applied plasmid DNA complexed to PEI followed by a mucosal protein boost generates sufficient antigen-specific humoral antibody production to protect from mucosal viral challenge.

Pulmonary delivery of DNA vaccine constructs using deacylated PEI elicits immune responses and protects against viral challenge infection.

Vaccination through mucosal surfaces has been shown to elicit antiviral immune responses against a number of mucosal pathogens. Here we demonstrate that both mucosal and systemic immune responses can be elicited against a model HIV-1 CN54gp140 antigen when cation-complexed plasmid DNA vaccines are applied topically to the murine pulmonary mucosa as an immune priming strategy. Furthermore, using an influenza challenge model we show that a plasmid DNA vaccine complexed to a less toxic form of PEI called dPEI (a nearly fully hydrolysed linear PEI with 11% additional free protonatable nitrogen atoms) can provide significant protection against a respiratory challenge infection in mice. Furthermore, we show that dPEI polyplexes have the potential to transfect not only mucosal epithelium, but also to enter deeper into tissues through the modulation of tight junction integrity. Taken together, these results demonstrate that less toxic forms of PEI can be effective delivery vehicles for plasmid DNAs to elicit cellular and humoral protective responses in vivo. Moreover, our observations suggest that these less toxic derivatives of PEI could be utilised for topical plasmid DNA vaccine delivery to human mucosal tissue surfaces, and that this application may permit dissemination of the immune responses through the linked mucosal network thus providing protective immunity at distal portals of pathogen entry.

Mucosal tissue tropism and dissemination of HIV-1 subtype B acute envelope-expressing chimeric virus.

Human immunodeficiency virus type 1 (HIV-1) transmission results from infection with one or a small number of variants from the donor quasispecies. Transmitted/founder (T/F) viruses have recently been identified from acutely infected patients, but the way in which they interact with primary targets of HIV-1 infection is poorly understood. We have conducted a biological characterization of a panel of subtype B T/F acute and chronic envelope (Env)-expressing chimeric virus in primary human target cells and mucosal tissues. Both acute and chronic Envs preferentially replicated in peripheral blood mononuclear cells (PBMC) and a CD4 T-cell line compared to monocyte-derived macrophages, or dendritic cells (DC). In a model of trans infection from monocyte-derived dendritic cells to T cells, chimeric virus from acute Envs achieved significantly lower titers compared to chronic Envs. Challenge of primary human mucosal tissues revealed significantly higher levels of replication in chronic Env-expressing virus in rectal tissue compared to cervical and penile tissues and enhanced replication in tonsillar tissue relative to acute Envs. In agreement with data from the DC to T-cell trans infection assay, chronic Env-chimeric virus pools were transmitted more efficiently by migratory cells from cervical and penile tissues to CD4(+) T cells than individual acute Env chimeras. These data indicate that virus with HIV-1 Envs of transmitted acute infections preferentially replicate in T cells rather than macrophages or dendritic cells and are less efficiently transmitted from antigen-presenting cells to CD4 T cells than chronic Envs. Such properties together with chemokine (C-C motif) receptor 5 (CCR5) use may confer an advantage for transmission.

Evaluation of CD62L expression as a marker for vaccine-elicited memory cytotoxic T lymphocytes.

The development of successful vaccination strategies for eliciting cytotoxic T lymphocytes (CTLs) will be facilitated by the definition of strategies for subdividing CTLs into functionally distinct subpopulations. We assessed whether surface expression of a number of cell-surface proteins could be used to define functionally distinct subpopulations of memory CTLs in mice immunized with a recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope (Env). We found changes in cell-surface expression of CD11a, CD44, CD45RB, CD49d, CD54 and CD62L on Env-specific CD8(+) T cells that appeared to differentiate them from other CD8(+) T cells within 1 week to 1 month following immunization. Further, we saw an up-regulation of CD62L surface expression on Env-specific CD8(+) memory T cells several months after immunization. However, CD62L expression did not correlate with differences in the abilities of CTLs to proliferate or produce interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) in vitro in response to Env peptide stimulation. Moreover, the expression of CD62L did not allow differentiation of CTLs into subpopulations with distinct expansion kinetics in vivo after adoptive transfer into naïve mice and subsequent boosting of these mice with a recombinant adenovirus expressing HIV-1 Env. Therefore, the definition of memory CD8(+) T-cell subpopulations on the basis of CD62L expression in mice does not allow the delineation of functionally distinct CTL subpopulations.

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.

Recruitment of different subsets of antigen-presenting cells selectively modulates DNA vaccine-elicited CD4+ and CD8+ T lymphocyte responses.

The immunogenicity of plasmid DNA vaccines may be limited by the availability of professional antigen-presenting cells (APC) at the site of inoculation. Here we demonstrate that the types of APC recruited to the injection site can selectively modulate CD4(+) or CD8(+) T lymphocyte responses elicited by an HIV-1 Env DNA vaccine in mice. Coadministration of plasmid GM-CSF with the DNA vaccine resulted in the recruitment of macrophages to the site of inoculation and specifically augmented vaccine-elicited CD4(+) T lymphocyte responses. In contrast, coadministration of plasmid MIP-1 alpha with the DNA vaccine resulted in the recruitment of dendritic cells to the injection site and enhanced vaccine-elicited CD8(+) T lymphocyte responses. Interestingly, coadministration of both plasmid GM-CSF and plasmid MIP-1 alpha with the DNA vaccine recruited both macrophages and dendritic cells and led to a synergistic and sustained augmentation of CD4(+)and CD8(+) T lymphocyte responses. These data demonstrate the critical importance of locally recruited professional APC in determining the magnitude and nature of immune responses elicited by plasmid DNA vaccines. Moreover, these studies show that different subsets of professional APC can selectively modulate DNA vaccine-elicited T lymphocyte responses.