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On March 24, 2017, more than 90 experts in blood safety and international development from blood centers, industry, government, and international and nongovernmental organizations gathered in Arlington, Virginia, for the Third International Blood Safety Forum, cosponsored by America's Blood Centers and Global Healing. This report summarizes presentations and major conclusions. The meeting explored ways to increase access to affordable, safe blood for low- and lower-middle-income countries (LMICs) in an era when funding from the US President's Emergency Plan for AIDS Relief (PEPFAR) and the Global Fund has been redirected from preventing the spread of human immunodeficiency virus (HIV) to diagnosing and treating the 25 million-plus people living with HIV in LMICs. More effective management systems must be developed to improve cost recovery for blood. While blood systems become more sustainable, continued investment is required to keep them operating. The traditional model of large grants from bilateral and multilateral donors will need to be supplemented (or replaced) with public-private partnerships and nongovernmental investment. A continued emphasis on quality is fundamental. Blood systems must build quality programs, based on accepted standards, including hospitals, clinics, and rural health care providers to ensure proper and safe use of blood. Proposals to resolve health care inequities between LMICs and high-income countries (HICs) must include helping LMICs to define sustainable national policies and practices for blood availability and utilization to suit local contexts. The blood safety lexicon should be revised to include availability, accessibility, and affordability of safe blood and blood products as the goal of all blood safety initiatives., (© 2018 AABB.)

Influenza virus causes acute upper and lower respiratory infections and is the most likely, among known pathogens, to cause a large epidemic in humans. Influenza virus mutates rapidly, enabling it to evade natural and vaccine-induced immunity. Furthermore, influenza viruses can cross from animals to humans, generating novel, potentially pandemic strains. Currently available influenza vaccines induce a strain specific response and may be ineffective against new influenza viruses. The difficulty in predicting circulating strains has frequently resulted in mismatch between the annual vaccine and circulating viruses. Low-resource countries remain mostly unprotected against seasonal influenza and are particularly vulnerable to future pandemics, in part, because investments in vaccine manufacturing and stockpiling are concentrated in high-resource countries. Antibodies that target conserved sites in the hemagglutinin stalk have been isolated from humans and shown to confer protection in animal models, suggesting that broadly protective immunity may be possible. Several innovative influenza vaccine candidates are currently in preclinical or early clinical development. New technologies include adjuvants, synthetic peptides, virus-like particles (VLPs), DNA vectors, messenger RNA, viral vectors, and attenuated or inactivated influenza viruses. Other approaches target the conserved exposed epitope of the surface exposed membrane matrix protein M2e. Well-conserved influenza proteins, such as nucleoprotein and matrix protein, are mainly targeted for developing strong cross-protective T cell responses. With multiple vaccine candidates moving along the testing and development pipeline, the field is steadily moving toward a product that is more potent, durable, and broadly protective than previously licensed vaccines., (Copyright © 2016 World Health Organization. Published by Elsevier Ltd.. All rights reserved.)

In anticipation of the successful eradication of wild polio virus, alternative vaccination strategies for public-sector markets of low-resource countries are extremely important, but are still under development. Following polio eradication, inactivated polio vaccine (IPV) would be the only polio vaccine available, and would be needed for early childhood immunization for several years, as maintenance of herd immunity will be important for sustaining polio eradication. Low-cost combination vaccines containing IPV could provide reliable and continuous immunization in the post-polio eradication period. Combination vaccines can potentially simplify complex pediatric routine immunization schedules, improve compliance, and reduce costs. Hexavalent vaccines containing Diphtheria (D), Tetanus (T), whole cell pertussis (wP), Hepatitis B (HBV), Haemophilus b (Hib) and the three IPV serotype antigens have been considered as the ultimate combination vaccine for routine immunization. This product review evaluates potential hexavalent vaccine candidates by composition, probable time to market, expected cost of goods, presentation, and technical feasibility and offers suggestions for development of low-cost hexavalent combination vaccines. Because there are significant technical challenges facing wP-based hexavalent vaccine development, this review also discusses other alternative approaches to hexavalent that could also ensure a timely and reliable supply of low-cost IPV based combination vaccines.

Background: A novel multicomponent vaccine against meningococcal capsular group B (MenB) disease contains four major components: factor-H-binding protein, neisserial heparin binding antigen, neisserial adhesin A, and outer-membrane vesicles derived from the strain NZ98/254. Because the public health effect of the vaccine, 4CMenB (Novartis Vaccines and Diagnostics, Siena, Italy), is unclear, we assessed the predicted strain coverage in Europe., Methods: We assessed invasive MenB strains isolated mainly in the most recent full epidemiological year in England and Wales, France, Germany, Italy, and Norway. Meningococcal antigen typing system (MATS) results were linked to multilocus sequence typing and antigen sequence data. To investigate whether generalisation of coverage applied to the rest of Europe, we also assessed isolates from the Czech Republic and Spain., Findings: 1052 strains collected from July, 2007, to June, 2008, were assessed from England and Wales, France, Germany, Italy, and Norway. All MenB strains contained at least one gene encoding a major antigen in the vaccine. MATS predicted that 78% of all MenB strains would be killed by postvaccination sera (95% CI 63-90, range of point estimates 73-87% in individual country panels). Half of all strains and 64% of covered strains could be targeted by bactericidal antibodies against more than one vaccine antigen. Results for the 108 isolates from the Czech Republic and 300 from Spain were consistent with those for the other countries., Interpretation: MATS analysis showed that a multicomponent vaccine could protect against a substantial proportion of invasive MenB strains isolated in Europe. Monitoring of antigen expression, however, will be needed in the future., Funding: Novartis Vaccines and Diagnostics., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or "relative potency" (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified.

Factor H binding protein (fHbp), one of the main antigens of new vaccines against serogroup B meningococcus, varies in amino acid sequence and level of expression in different clinical isolates. To evaluate the contribution of amino acid sequence variability to vaccine coverage, we constructed a strain that is susceptible to bactericidal killing only by anti-fHbp antibodies and engineered it to express equal levels of 10 different fHbp sub-variants from a constitutive promoter. Testing of these isogenic strains showed that sera from mice or adult volunteers vaccinated with fHbp variant 1.1 were bactericidal against all sub-variants 1 sequences, however the titer against the most distant sequences were several times lower. Sera from vaccinated infants were more susceptible to amino acid variations and they had lower or no bactericidal activity against the distant sub-variants 1 sequences in comparison with sera from adults given the same vaccines. The low coverage provided by fHbp could be overcome using a multicomponent vaccine. We conclude that fHbp is a very important antigen that induces bactericidal antibodies in animals, adults and infants. However, given its high variability of sequence and expression level, it is unlikely that fHbp alone can provide good protection in infants against the distant amino acid sequence variants and therefore multicomponent vaccines inducing protective immunity also against other antigens are more likely to induce a broad protective immunity in all age groups., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

GNA2132 is a Neisseria meningitidis antigen of unknown function, discovered by reverse vaccinology, which has been shown to induce bactericidal antibodies in animal models. Here we show that this antigen induces protective immunity in humans and it is recognized by sera of patients after meningococcal disease. The protein binds heparin in vitro through an Arg-rich region and this property correlates with increased survival of the unencapsulated bacterium in human serum. Furthermore, two proteases, the meningococcal NalP and human lactoferrin, cleave the protein upstream and downstream from the Arg-rich region, respectively. We conclude that GNA2132 is an important protective antigen of N. meningitidis and we propose to rename it, Neisserial Heparin Binding Antigen (NHBA).

The standard serological methods present limitations for the measurement of immunity against H5N1 influenza strains. The hemagglutination inhibition (HI) assay lacks sensitivity and requires standardization, while the viral micro-neutralization (MN) assay needs handling of live virus. We produced pseudoparticles expressing hemagglutinin from clades 1 or 2 H5N1 in order to measure neutralizing antibodies in human sera after prime-boost vaccination with plain or MF59-adjuvanted H5N1 clade 1 subunit vaccines. Titers measured by pseudoparticle neutralization (PPN) assay significantly correlated with those measured by HI, single radial haemolysis or MN, with a PPN titer of 1:357 corresponding to an MN titer of 1:80. Notably, results from the PPN assay, confirm that MF59-H5N1 vaccine induces potent and long-lasting neutralizing antibody responses not only against the vaccine strain, but also against several heterologous clade 2 strains. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of H5N1 vaccine immunogenicity.

Pandemic H1N1 influenza vaccine antigens are currently being manufactured. The MF(59) adjuvant has an established safety profile in humans and a proven ability to increase responses to some influenza vaccines in humans. To inform initial decisions on the use of these vaccine components to protect human populations, we have immunized mice with MF(59)-adjuvanted or non-adjuvanted pandemic influenza vaccine. Immunizing unprimed mice with a single dose of MF(59)-adjuvanted vaccine elicits functional antibody titers equivalent to those associated with protection of humans from seasonal influenza. Without adjuvant, two doses are required for a robust antibody response. Unadjuvanted vaccines with 0.5 and 1 microgram of antigen elicit equivalent titers. These data support including MF(59) in pandemic flu vaccines to rapidly protect young adults and children, who may have little or no previous exposure to influenza infection or immunization.

The immunologic and virologic outcome of therapeutic DNA-vaccines administered during antiretroviral therapy (ART) using electroporation with or without (interleukin) IL-2 treatment was evaluated in the SIVmac239/macaque model. Rhesus macaques inoculated with pathogenic SIVmac239 were treated with ART [(R(-9-(2-phosphonomethoxypropyl) adenine) (PMPA), FTC, Zerit] from weeks 13 to 41 postinfection (wpi). Group 1 (n = 7) received ART only, groups 2 and 3 (each n = 6) additionally received SIVmac239-derived gp140Env, GagPol, and TatRevNef plasmids by in vivo electroporation at 22, 26, 30, and 34 wpi, and group 3 also IL-2 for 14 days after each vaccination. Endpoints evaluated were viral load, Gag(181189)-specific CD8+ T-cell responses in MamuA01+ animals, lymphoproliferative responses, and CD4 T-cell counts. Viremia in all animals dropped below 200 RNA copies/ml during ART. Frequencies of Gag(181189)-specific CD8+ T cells prior to ART were detectable in all three groups (1.27-3.01%) and increased significantly (p < 0.01) postvaccination with maximum responses after the fourth immunization (0.2% versus 3.49-7.15%). Gag(181189)-specific CD8+ T-cell frequencies increased post-ART cessation in all groups and remained at significantly higher levels (p < 0.001) until the end of the study (75 wpi) in both groups of vaccinated animals. Lymphoproliferative responses were detected against Gag in a limited number of animals after vaccination and post-ART. However, plasma RNA viral loads rebounded after ART termination to similar levels in all three groups, but remained below 10(5) copies/ml until the end of the study, which could be a late effect of the triple drug therapy.

Vaccination strategies that can block or limit heterosexual human immunodeficiency virus (HIV) transmissions to local and systemic tissues are the goal of much research effort. Herein, in a mouse model, we aimed to determine whether the enhancement of antibody responses through mucosal and systemic immunizations, previously observed with protein-based vaccines, applies to immunizations with DNA- or RNA-based vectors. Intranasal (i.n.) followed by intramuscular (i.m.) immunizations (i.n./i.m.) with polylactide-coglycolide (PLG)-DNA microparticles encoding HIV-gag (PLG-DNA-gag) significantly enhanced serum antibody responses, compared with i.m., i.n. or i.m. followed by i.n. (i.m./i.n.) immunizations. Moreover, while i.n./i.m., i.n. or i.m./i.n. immunizations with PLG-DNA-gag resulted in genital tract antibody responses, i.m. immunizations alone failed to do so. Importantly, beta7-deficient mice developed local and systemic antibody responses following i.n./i.m. immunization, or immunization via any other route, similar to those of wild-type mice. To compare the DNA with an RNA delivery system, immunizations were performed with VEE/SIN-gag replicon particles, composed of Venezuelan equine encephalitis virus (VEE) replicon RNA and Sindbis surface structure (SIN). i.n./i.m., compared with any other immunizations, i.n./i.m. immunization with VEE/SIN-gag resulted in enhanced genital tract but not serum antibody responses. These data show for the first time that mucosal followed by systemic immunizations with gene delivery systems enhance B-cell responses independent of the mucosal homing receptors alpha4beta7 and alphaEbeta7.

Mucosal and systemic transmission of HIV is prevalent. Therefore, mucosal followed by parenteral immunizations with chimeric vs. complete alphavirus-based replicon particles, encoding an HIV envelope glycoprotein, were tested. Female rhesus macaques were immunized intranasally and then intramuscularly. Following the immunizations, enhanced mucosal and systemic antibody responses were detected with the chimeric compared to the complete replicon particles. Although similar proportions of the same peripheral blood monocyte lineage target cells were infected with the chimeric vs. the complete replicon particles, the latter resulted in enhanced expression of the gene of interest, suggesting a possible mechanism of the enhanced immunogenicity.

HIV envelope glycoprotein (Env) is the target for inducing neutralizing antibodies. Env is present on the virus surface as a trimer, and, upon binding to CD4, a cascade of events leads to structural rearrangement exposing the co-receptor binding site and entry into the CD4+ host target cells. We have designed monomeric and trimeric Env constructs with and without deletion of the variable loop 2 (ΔV2) from SF162, a subtype B primary isolate, and performed biophysical, biochemical and immunological studies to establish a potential structure–functional relationship. We expressed these Envs in CHO cells, purified the proteins to homogeneity and performed biophysical studies to define the binding properties to CD4, structural characteristics and exposure of epitopes recognized by b12 and CD4i mAb (17B) on both full-length and mutant HIV Env proteins. Parameters evaluated include oligomerization state, number and affinity of CD4 binding sites, enthalpy and entropy of the Env–CD4 interaction and affinity for b12 and 17b mAbs. We observed one CD4 binding site per monomer and three active CD4 binding sites per trimer. A40-fold difference in affinity of the gp120 monomer vs. the o-gp140 trimer towards CD4 was observed (Kd = 58 nM and 1.5 nM, respectively),whereas only a 2-fold difference was observed for the V2 deleted Envs (Kd of gp120ΔV2 = 19 nM, Kd of o-gp140DV2 = 9.3 nM). Monomers had 3-fold higher affinity to the mAb 17b and at least 3-fold weaker affinity to b12 compared to trimers, with gp120DV2 having the weakest affinity for b12 (Kd = 446 nM). Affinity of CD4 binding correlated with proportion of the antibodies induced against the conformational epitopes by the corresponding Envs, and changes in mAb binding correlated with the induction of antibodies directed against linear epitopes. Furthermore,biophysical analysis reveals that the V2 deletion has broad structural implications in the monomer not shared by the trimer, and these changes are reflected in the quality of the immune responses induced in rabbits. These data suggest that biophysical characteristics of HIV Env, such as affinity for CD4, and exposure of important neutralizing epitopes, such as those recognized by b12 mAb, may be important predictors of its in vivo efficacy and may serve as important surrogate markers for screening Env structures as potential vaccine candidates., (©2006 Elsevier Inc. All rights reserved.)

The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8(+) T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press) and support a subtype C in-frame gagpol fusion gene vaccine.

The urgent need for a vaccine against HIV/AIDS requires that multiple strategies be employed and evaluated in a clinical setting. V2-loop deleted trimeric envelope (Env) immunogens (protein and DNA) from subtypes B and C human immunodeficiency virus type 1 (HIV-1) strains were produced for ongoing and future clinical evaluations with other HIV antigens, adjuvants and deliveries.

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.

In the years following the publication of the initial in vivo demonstration of the ability of plasmid DNA to generate protective immune responses, DNA vaccines have entered into a variety of human clinical trials for vaccines against various infectious diseases and for therapies against cancer, and are in development for therapies against autoimmune diseases and allergy. They also have become a widely used laboratory tool for a variety of applications ranging from proteomics to understanding Ag presentation and cross-priming. Despite their rapid and widespread development and the commonplace usage of the term "DNA vaccines," however, the disappointing potency of the DNA vaccines in humans underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. This review will provide a brief background of DNA vaccines including the insights gained about the varied immunological mechanisms that play a role in their ability to generate immune responses.

Investigation into the mechanism of action of vaccine adjuvants provides opportunities to define basic immune principles underlying the induction of strong immune responses and insights useful for the rational development of subunit vaccines. A novel HIV vaccine composed of plasmid DNA-encoding p55 gag formulated with poly-lactide-co-glycolide microparticles (PLG) and cetyl trimethyl ammonium bromide (CTAB) elicits both serum antibody titers and cytotoxic lymphocyte activity in mice at doses two orders of magnitude lower than those required for comparable response to plasmid DNA in saline. Using this model, we demonstrated the increase in potency requires the DNA to be complexed to the PLG-CTAB microparticles. Furthermore, the PLG-CTAB-DNA formulation increased the persistence of DNA at the injection site, recruited mononuclear phagocytes to the site of injection, and activated a population of antigen presenting cells. Intramuscular immunization with the PLG-CTAB-DNA complex induced antigen expression at both the injection site and the draining lymph node. These findings demonstrate that the PLG-CTAB-DNA formulation exhibits multiple mechanisms of immunopotentiation.

Control of the worldwide AIDS pandemic may require not only preventive but also therapeutic immunization strategies. To meet this challenge, the next generation of human immunodeficiency virus type 1 (HIV-1) vaccines must stimulate broad and durable cellular immune responses to multiple HIV antigens. Results of both natural history studies and virus challenge studies with macaques indicate that responses to both Gag and Pol antigens are important for the control of viremia. Previously, we reported increased Rev-independent expression and improved immunogenicity of DNA vaccines encoding sequence-modified Gag derived from the HIV-1(SF2) strain (J. zur Megede, M. C. Chen, B. Doe, M. Schaefer, C. E. Greer, M. Selby, G. R. Otten, and S. W. Barnett, J. Virol. 74: 2628-2635, 2000). Here we describe results of expression and immunogenicity studies conducted with novel sequence-modified HIV-1(SF2) GagPol and Pol vaccine antigens. These Pol antigens contain deletions in the integrase coding region and were mutated in the reverse transcriptase (RT) coding region to remove potentially deleterious enzymatic activities. The resulting Pol sequences were used alone or in combination with sequence-modified Gag. In the latter, the natural translational frameshift between the Gag and Pol coding sequences was either retained or removed. Smaller, in-frame fusion gene cassettes expressing Gag plus RT or protease plus RT also were evaluated. Expression of Gag and Pol from GagPol fusion gene cassettes appeared to be reduced when the HIV protease was active. Therefore, additional constructs were evaluated in which mutations were introduced to attenuate or inactivate the protease activity. Nevertheless, when these constructs were delivered to mice as DNA vaccines, similar levels of CD8(+) T-cell responses to Gag and Pol epitopes were observed regardless of the level of protease activity. Overall, the cellular immune responses against Gag induced in mice immunized with multigenic gagpol plasmids were similar to those observed in mice immunized with the plasmid encoding Gag alone. Furthermore, all of the sequence-modified pol and gagpol plasmids expressed high levels of Pol-specific antigens in a Rev-independent fashion and were able to induce potent Pol-specific T- and B-cell responses in mice. These results support the inclusion of a gagpol in-frame fusion gene in future HIV vaccine approaches.

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1(US4). We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (R(h)) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller R(h) of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

This chapter describes a modified enzyme-linked immunosorbent assay (ELISA) employing assay conditions that ensure specificity of antibody binding and favor detection primarily of high-avidity serum IgG antibodies to meningococcal serogroup C polysaccharide (1). Antibody-binding assays such as the ELISA offer a convenient and reproducible method for quantifying anticapsular antibody responses to vaccination or disease. However, the results do not always correlate well with assays of antibody functional activity, such as bactericidal activity. For example, a standardized ELISA for measurement of antibody responses to meningococcal C polysaccharide has been described (2). When used to assay relatively homogenous populations of serum antibodies, for example in sera from immunized adults (3), or from younger individuals given a single vaccine (4), the results of the standard ELISA correlated well with serum bactericidal titers. In contrast, much lower correlations were observed when assaying heterologous populations of serum antibodies from vaccinated infants or toddlers (5,6), particularly from clinical studies comparing antibody responses of different age groups (6), or studies comparing responses to a polysaccharide and conjugate vaccines (7,8).

Dendritic cells (DC) play a key role in antigen presentation and activation of specific immunity. Much current research focuses on harnessing the potency of DC for vaccines, gene therapy, and cancer immunotherapy applications. However, DC are not readily transfected in vitro by traditional nonviral techniques. A novel DNA vaccine formulation was used to determine if DC are transfected in vitro. The formulation consists of plasmid DNA adsorbed on to cationic microparticles composed of the biodegradable polymer polylactide-co-glycolide (PLG) and the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Using preparations of fluorescent-labeled plasmid DNA formulated on PLG-CTAB microparticles to study internalization by macrophages and dendritic cells in vitro and in vivo, we found that most, but not all, of the fluorescence was concentrated in endosomal compartments. Furthermore, uptake of plasmid DNA encoding HIV p55 gag adsorbed to PLG-CTAB microparticles by murine bone marrow-derived dendritic cells resulted in target gene expression, as detected by RT-PCR. The antigen was subsequently processed and presented, resulting in stimulation of an H-2kd-restricted, gag-specific T cell hybridoma. Activation of the hybridoma, detected by IL-2 production, was dose-dependent in the range of 0.1-20 microg DNA (10-2000 microg PLG) and was sustained up to 5 days after transfection. Thus, adsorption of plasmid DNA on PLG-CTAB microparticles provides a potentially useful nonviral approach for in vitro transfection of dendritic cells. Gene Therapy (2000) 7, 2105-2112.

There is reasonable evidence that both cross-priming and direct transfection of antigen-presenting cells (APCs) play a role in induction of immune responses by DNA vaccines. It is not known which mode is more important for priming cytotoxic T cell responses, but both are sufficient and neither alone is necessary. Hence, a rational strategy for increasing DNA vaccine potency would be to facilitate both pathways. With regard to cross-priming, a better understanding of the nature of the antigen transferred and the molecules/cells involved may suggest ways to design DNA vaccines to enhance this pathway. With respect to transfection of APCs, certain DNA formulations or delivery systems may be able to target APCs for increased DNA uptake. Other considerations include recruitment of APCs to the site of DNA injection and manipulation of these cells to ensure the proper activation state for priming immune responses. The burgeoning scientific literature in these areas indicates that much effort is currently being directed toward these goals.

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-microl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 microl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-microl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

A pneumococcal conjugate vaccine (PCV) has been developed consisting of capsular polysaccharide (Ps) coupled to the outer membrane protein complex of Neiserria meningitidis serogroup B. Experiments were conducted in infant rhesus monkeys to assess the potential to administer multiple Pn types in a single vaccine. A single type conjugate, 6B, was dosed from 0.025 to 25 microg Ps. Peak anti-6B Ps Ab titers were seen at lower doses of 0.025 and 0.25 microg Ps, while reduced titers of anti-6B Ps Ab were observed at the highest doses of conjugate administered, 2.5 and 25 microg Ps. By mixing free Ps, carrier, or another monovalent PCV with this 6B PCV, it was determined that reduced anti-6B Ps titers at high PCV doses were associated only with the quantity of type-specific Ps in the conjugate. Thus, increasing the amount of carrier protein or adding an additional monovalent conjugate did not significantly affect the response to type 6B Ps. These results suggest that, given an appropriately determined dose per individual pneumococcal Ps type, a multivalent PCV that includes many different types should have satisfactory clinical immunogenicity.

The immunogenicity and protective efficacy of DNA vaccines have been amply demonstrated in numerous animal models of infectious disease. However, the feasibility of DNA vaccines for human use is not yet known. In order to investigate potential means of increasing the potency of DNA vaccines, conventional adjuvants such as aluminum salts were tested. Coadministration of these adjuvants with DNA vaccines substantially enhanced the ability of these vaccines to induce antibody responses up to 100-fold in mice and guinea pigs, and 5-10-fold in non-human primates. Effective formulations had no demonstrable effect on the levels of antigen expression in situ and consisted of adjuvants that did not form complexes with the plasmid DNA; rather they exerted their effects on antigen after expression in situ. Therefore, the potency of DNA vaccines both in laboratory rodents and in non-human primates can be substantially increased by simple formulation with conventional aluminum adjuvants.

Intramuscular injection of BALB/c mice with a DNA plasmid encoding nucleoprotein (NP) from influenza virus A/PR/8/34 (H1N1) provides cross-strain protection against lethal challenge with influenza virus A/HK/68 (H3N2). CTL specific for the H-2Kd-restricted epitope NP147-155 are present in these mice and are thought to play a role in the protection. To assess the effectiveness of NP DNA immunization in comparison with influenza virus infection in the induction of CTL responses, we monitored the frequency of CTL precursors (CTLp) in mice following i.m. injection with NP DNA or intranasal infection with influenza virus and showed that the CTLp frequency in NP DNA-immunized mice can reach levels found in mice that had been infected with influenza virus. We also measured the CTLp frequency, anti-NP Ab titers, and T cell proliferative responses in mice that were injected with titrated dosages of NP DNA and documented a correlation of the CTLp frequency and the Ab titers, but not proliferative responses, with the injection dose. Furthermore, we observed a positive correlation between the frequency of NP147-155 epitope-specific CTLp and the extent of protective immunity against cross-strain influenza challenge induced by NP DNA injection. Collectively, these results and our early observations from adoptive transfer experiments of in vitro activated lymphocytes from NP DNA-immunized mice suggest a protective function of NP-specific CTLp in mice against cross-strain influenza virus challenge.

DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL) responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2Kd-restricted NP147-155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 microgram) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquintination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.

Influenza is a leading cause of morbidity and mortality in older persons. The current influenza vaccine is only modestly successful, in part because of an age-related decline in immunogenicity and also because it induces only type-specified immunity. To overcome this, we evaluated DNA vaccines encoding A/PR8/34 haemagglutinin (HA) and nucleoprotein (NP) in young and aged BALB/c mice. Control mice were given formalin-inactivated A/PR8/34, control DNA, or a non-lethal dose of PR8. Aged mice given HA DNA developed slightly lower anti-HA serum antibodies than young mice; however, both young and aged mice were protected from a homotypic PR8 challenge. Following vaccination with NP DNA, both young and aged mice developed anti-NP bulk cytotoxic T-lymphocyte (CTL) activity and pCTL frequency similar to control animals. When challenged with a low dose of A/HK/68 (H3N2) influenza virus, both young mice and aged mice showed significant protection as measured by inhibition of weight loss. When challenged with a relatively high dose of A/HR/68 (H3N2) influenza virus, however, the anti-NP vaccine only partially protected young mice and failed to protect aged mice. These data demonstrate that DNA-based vaccines are immunogenic in aged animals, but suggest that factors other than the age-related decline in CTL activity also contribute to the increased morbidity and mortality of influenza in the elderly.

Streptococcus pneumoniae is responsible for high rates of pneumococcal bacteremia, meningitis, pneumonia, and acute otitis media worldwide. Protection from disease is conferred by antibodies specific for the polysaccharide (Ps) capsule of the bacteria. Of the four types of group 9 pneumococci, types 9N and 9V cause the most disease, and both types are included in the polyvalent pneumococcal vaccine. The type 9V capsule consists of repeating pentasaccharide units linearly arranged, with an average of 1 to 2 mol of O-acetate side chains per mol of repeat units, added in a complex pattern in which not all repeat units are alike. alpha-GlcA residues may be O-acetylated in the 2 (17%) or 3 (25%) position and beta-ManNAc residues may be O-acetylated in the 4 (6%) or 6 (55%) position. Under certain conditions, the O-acetate side chains are subject to oxidation, which results in subsequent de-O-acetylation of a significant number of the repeat units. This de-O-acetylation could adversely affect the efficacy of a vaccine containing the 9V Ps. A study was undertaken to compare the relative contributions of O-acetate and Ps backbone epitopes in the immune response to S. pneumoniae 9V type-specific Ps. In both an infant rhesus monkey model and humans, antibodies against the non-O-acetylated 9V backbone as well as against O-acetylated 9V Ps were detected. Functional (opsonophagocytic) activity was observed in antisera in which the predominant species of antibody recognized de-O-acetylated 9V Ps. We concluded that the O-acetate side groups, while recognized, are not essential to the ability of the 9V Ps to induce functional antibody responses.

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745-1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.

Immunization with plasmid DNA encoding antigenic proteins elicits both antibody and cell-mediated immune responses. This method of producing the protein antigens of interest directly in host cells can provide appropriate tertiary structure for the induction of conformationally specific antibodies, and also facilitates the induction of cellular immune responses. DNA immunization has provided effective protective immunity in various animal models. The immune responses induced by DNA vaccines may in some instances be preferable to those produced by immunization using conventional methods. DNA vaccination appears to be applicable to a variety of pathogens and is a useful method of raising immune responses. Thus this approach to vaccination has the potential to be a successful method of rapidly screening for antigens capable of inducing protective immunity, and of inducing protective immunity against pathogens of clinical importance.

Previously we showed that immunization of ferrets with DNA encoding the hemagglutinin (HA), nucleoprotein (NP), and matrix protein (M1) of influenza virus induced protective immune responses. A DNA vaccine encoding HA (from a 1991 strain), NP and M1 (from a 1989 strain) protected ferrets better against challenge with the antigenic drift variant A/Georgia/03/93 than did the inactivated vaccine from the 1992-93 influenza season. Here we report that the same DNA vaccine protected ferrets against a second, further divergent, drift variant (A/Johannesburg/33/94). Furthermore, the extent of protection provided by the DNA vaccine was equivalent to the homologous protection provided by an inactivated vaccine that exactly matched the challenge strain.

Intramuscular injection of plasmid DNA expression vectors results in transfection of myocytes in situ. To determine whether expression of antigen by myocytes is sufficient to induce protective cell-mediated immunity, stably transfected myoblasts expressing influenza nucleoprotein (NP) were transplanted into mice. These animals produced high-titer anti-NP antibodies and MHC class I-restricted cytotoxic T lymphocytes, and were protected from a cross-strain lethal challenge with influenza A virus. Therefore, antigen expression by muscle cells in vivo is sufficient to confer protective cell-mediated immunity.

Background: MHC class I molecule-restricted cytotoxic T-lymphocyte (CTL) responses are induced following either intramuscular (i.m.) injection of a DNA plasmid encoding influenza virus nucleoprotein (NP) or transplantation of myoblasts stably transfected with the NP gene, the latter indicating that synthesis of NP by myocytes in vivo is sufficient to induce CTL. The present study was designed to investigate the role of muscle cells and involvement of professional antigen-presenting cells (APCs) in priming CTL responses following DNA vaccination., Materials and Methods: Parent-->F1 bone marrow (BM) chimeric mice were generated whose somatic cells include muscle cells bearing both parental MHC haplotypes, while their professional APCs express only the donor MHC haplotypes., Results and Conclusions: Upon injection of NP DNA, or after infection with influenza virus, CTL responses generated in the chimeras were restricted to the donor MHC haplotype. Thus cells of BM lineage were definitively shown to be responsible for priming such CTL responses after infection or DNA immunization. Moreover, expression of antigen by muscle cells in BM chimeric mice after myoblast transplantation is sufficient to induce CTL restricted only by the MHC haplotype of the donor BM. This indicates that transfer of antigen from myocytes to professional APCs can occur, thus obviating a requirement for direct transfection of BM-derived cells.

DNA immunization offers a novel means to induce cellular immunity in a population with a heterogeneous genetic background. An immunorecessive cytotoxic T-lymphocyte (CTL) epitope in influenza virus nucleoprotein (NP), residues 218 to 226, was identified when mice were immunized with a plasmid DNA encoding a full-length mutant NP in which the anchor residues for the immunodominant NP147-155 epitope were altered. Mice immunized with wild-type or mutant NP DNA were protected from lethal cross-strain virus challenge, and the protection could be adoptively transferred by immune splenocytes, indicating the role of cell-mediated immunity in the protection. DNA immunization is capable of eliciting protective cellular immunity against both immunodominant and immunorecessive CTL epitopes in the hierarchy seen with virus infection.

DNA vaccination is an effective means of inducing both humoral and cell-mediated immunity in animal models of infectious disease. Presented here are data generated in two distinct disease models; one viral (influenza) and one bacterial (tuberculosis). Specifically, plasmid DNA encoding an influenza virus antigen (nucleoprotein; NP) and a Mycobacterium tuberculosis antigen (antigen 85; Ag85) were prepared and tested as DNA vaccines in mice. In both cases, high titer antibody responses and robust cell-mediated immune responses were induced against the respective antigens. With respect to the latter, lymphocyte proliferation, Th1-type cytokine secretion, and cytotoxic T lymphocyte responses were observed upon restimulation with antigen in vitro. Furthermore, protective efficacy in animal challenge models was demonstrated in both systems. The data support the hypothesis that DNA vaccination will prove to be a broadly applicable technique for inducing immunity against various infectious diseases.

The development of improved methods for the production of vaccines have fostered the use of purified subunits in place of live attenuated and whole inactivated organisms. Greater understanding of immune mechanisms has stimulated efforts to design vaccines that elicit specific mechanisms of resistance to particular pathogens. Together these forces have driven a search for novel adjuvants with specific properties optimally suited to vaccines for particular diseases. At the First International Conference on Immunology and Aging, a mini-symposium was devoted to the topic of new adjuvants. This review and the following two papers summarize the proceedings of the mini-symposium and illustrate the potential for the development of new adjuvants of greater efficacy and specificity, and the complexities involved in bringing new discoveries in adjuvants from the laboratory to the clinic.

Immunization with plasmid DNA encoding antigenic proteins elicits both antibody and cell-mediated immune responses. This method of producing the protein antigens of interest directly in host cells can provide appropriate tertiary structure for the induction of conformationally specific antibodies, and also facilitates the induction of cellular immune responses. DNA immunization has provided effective protective immunity in various animal models. The immune responses induced by DNA vaccines may in some instances be preferable to those produced by immunization using conventional methods. DNA vaccination appears to be applicable to a variety of pathogens and is a useful method of raising immune responses. Thus this approach to vaccination has the potential to be a successful method of rapidly screening for antigens capable of inducing protective immunity, and of inducing protective immunity against pathogens of clinical importance.

We have examined in detail the characteristics of the humoral immune response and protective efficacy induced by an influenza hemagglutinin (HA) DNA vaccine. In mice injected intramuscularly with HA DNA, the magnitude of the immune responses generated, as measured by ELISA and hemagglutination inhibiting (HI) antibodies, was directly related to the amount of DNA injected and the number of doses administered. The level of anti-HA antibodies in DNA-vaccinated mice was higher than that in convalescent immune mice and was maintained for at least 1.5 years. The immunoglobulin isotype profile of the antibodies was predominantly IgG2a, similar to that induced by live virus infection but in contrast to the relative abundance of IgG1 antibodies observed after inoculation with formalin-inactivated whole virus. The presence of pre-challenge HI antibodies was found to be a good correlate of protection, in that every animal with a detectable HI titer was protected from a lethal challenge. Complete protection from a lethal dose of influenza virus (A/PR/34), as judged by 100% survival and no weight loss, was conferred by as little as 1 microgram of DNA (given twice). Furthermore, mice injected with 10 to 100 micrograms doses, when subsequently challenged with virus, showed no increase in HI titer and no production of antibodies directed against the challenge virus, suggesting a substantial inhibition of virus replication after challenge.

In just a few years, injection of plasmid DNA to elicit immune responses in vivo has developed from an interesting observation to a viable vaccine strategy. DNA vaccines have been shown to elicit both cellular and humoral immune responses and to be effective in a variety of preclinical bacterial, viral, and parasitic animal models. This review will discuss the current knowledge of vector design, methods of plasmid delivery, immune responses elicited by various DNA vaccines, safety issues, and production and release of plasmid as a vaccine product. The potential of this new vaccine strategy and its future prospects is summarized.

DNA vaccination has proved to be a generally applicable technology in various preclinical animal models of infectious and noninfectious disease and several DNA vaccines have now entered phase I human clinical trials. It is too early to predict the effectiveness of DNA vaccines in humans and whether improved formulations of DNA vaccines will be required but several lines of investigation have suggested ways in which DNA vaccines may be improved, such as increases in expression, facilitation of DNA targeting or uptake, and enhancement of immune responses.

Genital infections with human papillomavirus (HPV) are increasingly recognized as a significant source of human disease; HPV is now implicated in up to 90% of cervical carcinomas. Neutralizing antibodies against papillomaviruses recognize conformational epitopes formed when viral capsid proteins assemble into virions or virus-like particles. Immunization with plasmid DNA encoding the major viral capsid protein L1 was studied as a means of inducing neutralizing antibodies and protection against virus challenge. In a cottontail rabbit papillomavirus (CRPV) model, immunization with plasmid DNA encoding L1 elicited conformationally specific neutralizing antibodies and provided immunity against papilloma formation upon challenge with CRPV. Immunization with DNA encoding the capsid protein may provide a means of protecting humans against HPV and would simplify the production of multivalent vaccines by combining plasmids that encode the viral capsid proteins of different strains. This may be of importance given the multiplicity of HPV types capable of causing disease.

The use of DNA and mRNA as vectors for immunization is a relatively recent development in the field of vaccines. The first paper demonstrating the efficacy of a DNA vaccine in an animal model of viral disease was published in 1993 (1). The rationale for using nucleic acids as vaccines came from the Initial observations that mtramuscular (im) injection of nonrephcating plasmid DNA expression vectors or mFWA-encoding reporter genes could result in the in vivo expression of proteins in mouse muscle cells (2). This ability to express proteins in vivo offers the opportunity to generate immune responses against foreign antigens encoded by the nucleic acid. In addition, both humoral and cell-mediated immune (CMI) responses, such as cytotoxic T-lymphocytes (CTL), can be induced. In general, CTL responses require endogenous expression of the antigen, such as during immunization with live viruses or replicating vectors, whereas subunit protein, polysaccharide conjugate, or inactivated virus vaccines generate humoral immune responses, but not CTL. Therefore, the technique of DNA injection has potential advantages over certain other vaccine technologies.