Microfluidization is an established technique for preparing emulsion adjuvant formulations for use in vaccines. Although this technique reproducibly yields high-quality stable emulsions, it is complex, expensive, and requires proprietary equipment. For this study, we developed a novel and simple low shear process to prepare stable reproducible emulsions without the use of any proprietary equipment. We found this process can produce a wide range of differently sized emulsions based on the modification of ratios of oil and surfactants. Using this process, we prepared a novel 20-nm-sized emulsion that was stable, reproducible, and showed adjuvant effects. During evaluation of this emulsion, we studied a range of emulsions with the same composition all sized below 200; 20, 90, and 160 nm in vivo and established a correlation between adjuvant size and immune responses. Our studies indicate that 160-nm-sized emulsions generate the strongest immune responses.

Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.

Parvovirus B19 is the causative agent of fifth disease in children, aplastic crisis in those with blood dyscrasias, and hydrops fetalis. Previous parvovirus B19 virus-like-particle (VLP) vaccine candidates were produced by co-infection of insect cells with two baculoviruses, one expressing wild-type VP1 and the other expressing VP2. In humans, the VLPs were immunogenic but reactogenic. We have developed new VLP-based parvovirus B19 vaccine candidates, produced by co-expressing VP2 and either wild-type VP1 or phospholipase-negative VP1 in a regulated ratio from a single plasmid in Saccharomyces cerevisiae. These VLPs are expressed efficiently, are very homogeneous, and can be highly purified. Although VP2 alone can form VLPs, in mouse immunizations, VP1 and the adjuvant MF59 are required to elicit a neutralizing response. Wild-type VLPs and those with phospholipase-negative VP1 are equivalently potent. The purity, homogeneity, yeast origin, and lack of phospholipase activity of these VLPs address potential causes of previously observed reactogenicity.

A plasmid DNA vaccine containing a fusion gene consisting of an HIV-1 subtype C gag and a modified subtype C pol was compared to a mixture of gag plus pol or gag plus HIV env plasmids. Plasmid DNA was delivered by intramuscular injection followed by electroporation in vivo. Two vaccinations were sufficient to induce high levels of Gag- and Pol-specific CD4 and CD8 T cells in peripheral blood. The gag-pol fusion plasmid was as immunogenic as the plasmid mixtures. Thus, DNA vaccination by intramuscular electroporation was an effective means for inducing high levels of Gag- and Pol-specific T cells, and a single gag-pol fusion DNA vaccine was sufficient for eliciting immune responses against both antigens.

Background Missed diagnoses of acute myocardial infarction (AM 1) in,the ambulatory setting can cause patient suffering and malpractice litigation. Multiple algorithms have been developed to detect the presence of coronary heart disease (CHD) or acute coronary ischemia.Methods We performed a case-control study of patients with no prior history of CHD presenting to outpatient practices with potential cardiac ischemia. Malpractice claims files were used to identify 18 cases of patients with missed AMls. For each case, we identified 3 control patients who had office visits for chest pain during the same month and assessed the association of 4 different prediction tools with missed AMI.Results The 18 cases of missed AMI had a 39% 1-month mortality rate. Cases were more likely than controls to be men (67% vs 26%, P =.001), to be smokers (88% vs 39% P <.001), and to have low HDL cholesterol (39 mg/dL vs 59 mg/dL, P <.001) and elevated total cholesterol (236 mg/dL vs 213 mg/dL, P =.01). A Framingham risk score predicting a 10-year risk of CHD greater than or equal to 10% and a positive score using the Goldman risk predictor were associated with an increased risk of missed AMI (odds ratio 5.7, 95% Cl 1.8-18.4 for Framingham risk score; odds ratio 7.2, 95% Cl 1.4-36.8 for Goldman risk predictor).Conclusions Among ambulatory patients with possible cardiac ischemia and no prior CHD, multiple algorithms may be useful for improvement of risk stratification.

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.

The potency of an HIV DNA vaccine was enhanced in rhesus macaques by in vivo electroporation, as judged by increased onset, magnitude and duration of antibody and cell-mediated immune responses against both components of a combination Gag and Env vaccine. These data demonstrate the utility of the electroporation technology for use in large animals.

Several vaccine technologies were evaluated for their abilities to induce anti-human immunodeficiency virus Gag immune responses in rhesus macaques. While no vaccine alone was able to induce broad and strong immune responses, these were achieved by priming with Gag DNA and boosting with Gag protein adsorbed to polylactide coglycolide microparticles. This regimen elicited strong antibodies, helper T cells, and cytotoxic T lymphocytes and thus holds promise as an effective vaccination scheme.

ABSTRACTThe effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIVgagDNA, adsorption onto PLG increased CD8+T-cell and antibody responses by ∼100- and ∼1,000-fold, respectively. In guinea pigs immunized with HIVenvDNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIVenvand HIVgagDNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of theenvDNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.

ABSTRACT A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1 SF2 p55 Gag were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55 Gag protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55 Gag and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gagprotease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8 + cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.

DNA vaccines can prime broad-based immune responses in small animal models. In the present study, we sought to evaluate the relative ability of DNA vaccines to induce humoral and cellular immune responses. Using a DNA vaccine encoding HIV gag in mice, we observed that CD8+ T cell responses were primed more readily than were antibody responses, particularly at low doses of DNA. These CD8+ T cell responses were detected in spleen cells, as well as at local sites such as the lung and draining lymph nodes. The potency of the HIV gag DNA vaccine used was sufficient to prime strong CTL responses in macaques, but only low to undetectable antibody responses. Therefore, DNA vaccines appear able to prime strong, broad CTL but only modest antibody responses. These results may have implications on the development of vaccines against infectious diseases where both CTL and antibody responses are desired, such as HIV.

Transferring small doses of T cells to heavily irradiated F1 mice expressing isolated MHC class I or class II differences invariably leads to rapid death from graft-vs-host disease (GVHD). Paradoxically, GVHD is mild or absent when irradiated F1 mice are reconstituted with large doses of unseparated parental strain spleen cells. This applies when bulk populations of B6 spleen cells are transferred to irradiated class II-different (B6 x bm12)F1 mice or class I-different (B6 x bm1)F1 mice. In this study, we examined whether the donor T cells in long-term spleen chimeras become tolerant to host MHC Ags. On the basis of skin-allograft rejection and induction of GVHD on adoptive transfer, the results show that the donor T cells display strong tolerance to host antigens; this applies to CD4+ cells in class II-different chimeras and to CD8+ cells in class I-different chimeras. In marked contrast to the profound tolerance seen by these in vivo parameters, little or no tolerance is observed in standard in vitro assays. The results illustrate that typical in vitro tests for alloreactivity are an imprecise guide to physiologic tolerance of T cells in vivo.

To seek information on the potential lifespan of normal B and T lymphocytes, lymph node (LN) cells from unprimed mice were transferred to H-2-identical severe combined immunodeficiency (SCID) hosts. At a population level, the donor B and T cells survived for at least 10 mo post-transfer with no reduction in their numbers. In terms of antibody production, LN-injected SCID mice remained responsive to several different antigens and contained unprimed precursors of memory cells for greater than or equal to 6 mo post-transfer. Most of the B and T cells recovered from LN-injected SCID mice expressed the typical virgin phenotype of mature lymphocytes from young mice. These findings suggest that many of the transferred lymphocytes might have remained in interphase as virgin cells from the time of injection. This did not apply to all of the transferred cells, however, because 20-40% of CD4+ cells from long-term SCID hosts displayed a memory phenotype, 7% incorporated 2-bromodeoxyuridine over 5 d, and total numbers of B and T cells increased gradually (twofold) over a 10-mo period. Collectively, the data favor the view that the pool of mature B and T cells in adult mice is largely self sufficient: some of the cells proliferate, presumably in response to environmental antigens, but many mature cells can remain quiescent for prolonged periods. Input of new cells from the primary lymphoid organs continues, but at a much reduced rate relative to young life.

Detailed information was sought on the capacity of purified B6 L3T4+ cells to elicit lethal graft-versus-host disease (GVHD) in irradiated class II-different class I-identical (C57BL/6 (B6) x bm 12)F1 hosts. When B6 L3T4+ cells were transferred in small doses (10(5) to 10(6) together with donor bone marrow (BM) cells, the recipients all developed acute lethal GVHD and most of the mice died within 2 wk, probably from gut damage; this syndrome was conspicuous only in mice treated with very heavy irradiation, i.e., 1000 rad. In marked contrast to L3T4+ cells given in small doses, transfer of large doses of B6 L3T4+ cells to heavily irradiated (B6 x bm 12)F1 hosts paradoxically resulted in only limited mortality: most of the recipients survived for greater than 6 mo and manifested little or no evidence of ill health. It is suggested that the capacity of large doses of L3T4+ cells to protect mice against lethal GVHD is a reflection of T helper function: the cellular immunity provided by the donor L3T4+ cells enables the host to repel pathogens entering through damaged mucosal surfaces, with the result that GVHD becomes sublethal. The protective function of L3T4+ cells in the B6----bm 12 combination was only seen in hosts given donor BM. With transfer of donor L3T4+ cells plus host BM, even lightly irradiated recipients died rapidly from hemopoietic failure. Because this syndrome failed to occur in mice given a mixture of donor and host BM, it would appear that L3T4+ cells destroyed host lymphohemopoietic cells by direct cytotoxicity rather than via a bystander effect.

Information was sought on the antigen-presenting cells (APC) required for stimulating primary mixed-lymphocyte reactions (MLR) by unprimed Lyt-2+ cells in the absence of added lymphokines. Like L3T4+ cells, Lyt-2+ cells gave very high MLR in response to H-2-different dendritic cells (DC). Surprisingly, high MLR were also elicited by thioglycollate-induced peritoneal exudate cells (PEC), including Ia- PEC; these cells were non-immunogenic for L3T4+ cells. Since PEC consisted almost entirely of macrophages (M phi), the data suggest that at least two different cell types, DC and M phi, can express APC function for unprimed Lyt-2+ cells. Since resident peritoneal M phi and in vitro cultured PEC were poorly immunogenic, the APC function of M phi might be limited to a subset of these cells, e.g. to immature M phi.

The triggering requirements of T cells differ for primed and unprimed cells: primed T cells can be triggered to produce lymphokines without viable antigen-presenting cells (APCs), apparently by crosslinking the T-cell receptor (TCR). Unprimed T cells do, however, require viable APCs and here Jonathan Sprent and Mary Schaefer review what type of cells can carry out this function, with particular reference to APCs for unprimed CD8+ cells.