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Biomedical subjects

David C Kaslow

Publications and source records attributed to David C Kaslow.

At least 19 recordsLinked to original sources

Biological feasibility of developing prophylactic vaccines for viral pathogens: incubation period as a critical parameter.

Vaccines for sixteen major human viral pathogens have been approved for use in humans. For most other major human viral pathogens, the absence of an efficacious prophylactic vaccine is not for lack of effort. To identify the key parameters of biologic feasibility that determine "certainty-of-success" for developing prophylactic vaccines for major human viral pathogens, basic characteristics of viruses, such as primary genomic structure, route of transmission, and durability of viral infection were reviewed. These basic characteristics of viruses simply do not associate with successful vaccine development, so one must look elsewhere for characteristics predictive of successful vaccine development. Herein existing data are put forth in a way that demonstrates the impact of the combined effect of incubation period and broadly protective, relative immunogenicity on the "certainty-of-success" of developing effective vaccines for thirty viral pathogens. Both incubation period and broadly protective, relative immunogenicity appear to have accounted for much of the past successes in vaccine development, and may be useful in predicting where near term successes are most apt to occur.

Antibodies, Viral↗

Qualification and performance characteristics of a quantitative enzyme-linked immunosorbent assay for human lgG antibodies to anthrax lethal factor antigen.

The contribution of Bacillus anthracis lethal factor (LF)-specific immune responses to protection against anthrax disease in humans remains incompletely defined due, in part, to a paucity of qualified reagents and a lack of standardized serological assays. Toward this end, we have identified and characterized suitable positive quality control and standard reference sera and developed, optimized, and qualified an enzyme-linked immunosorbent assay (ELISA) to measure LF-binding IgG. Herein we describe the performance characteristics of this ELISA and propose criteria for its use in the detection and quantification of anti-LF IgG in human serum.

Anthrax↗

Comparison of rabbit and mouse models for persistence analysis of plasmid-based vaccines.

Experiments were conducted with a cationic lipid-formulated pDNA vaccine (VCL-AB01) to evaluate the models used to determine biodistribution, persistence and the potential for integration (into genomic DNA) of plasmid DNA-based vaccines. Mice were injected with a high-dose volume of 50 microL unilaterally containing approximately 1.33 x 10(13) plasmid copy numbers (PCN) or a low-dose volume of 20 microL bilaterally ( approximately 5.3 x 10(12) PCN). Rabbits were injected bilaterally with a 0.5 mL ( approximately 1.33 x 10(14) PCN) volume. Injection site muscle tissue was harvested two days, one month, and two months postinjection for the low-dose murine and rabbit models and two days and two months postinjection for the high-dose murine model. Total DNA was extracted and analyzed by real-time quantitative PCR for sequences specific to the injected pDNA. The geometric mean PCN/microg of total DNA from the high and low dose models were compared to determine if injection volume impacts clearance and/or persistence. Results from these studies showed that PCN clearance over two months was similar in mice injected with 20 microL and rabbits injected with 0.5 mL, but PCN clearance was slower in mice injected with similar PCN in 50 microL (1.33 x 10(13) PCN) compared to 20 microL (5.3 x 10(12) PCN). Persistence at two months in the rabbit and low-dose murine models was comparable, with geometric mean of 5.22 x 10(3) PCN/microg of total DNA for the low-dose volume murine model and 2.81 x 10(3)/microg DNA for the rabbit model. Interanimal variability in persistence was not impacted by dose volume.

Animals↗

Vaxfectin enhances immunogenicity and protective efficacy of P. yoelii circumsporozoite DNA vaccines.

We evaluated the capacity of the cationic lipid based formulation, Vaxfectin, to enhance the immunogenicity and protective efficacy of DNA-based vaccine regimens in the Plasmodium yoelii murine malaria model. We immunized Balb/c mice with varying doses (0.4-50 microg) of plasmid DNA (pDNA) encoding the P. yoelii circumsporozoite protein (PyCSP), either in a homologous DNA/DNA regimen (D-D) or a heterologous prime-boost DNA-poxvirus regimen (D-V). At the lowest pDNA doses, Vaxfectin substantially enhanced IFA titers, ELISPOT frequencies, and protective efficacy. Clinical trials of pDNA vaccines have often used low pDNA doses based on a per kilogram weight basis. Formulation of pDNA vaccines in Vaxfectin may improve their potency in human clinical trials.

Adjuvants, Immunologic↗

I. Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine: evaluation of plasmid DNA biodistribution/persistence and integration.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

Animals↗

II. Cationic lipid-formulated plasmid DNA-based Bacillus anthracis vaccine: evaluation of plasmid DNA persistence and integration potential.

Several formulated plasmid DNA (pDNA)-based vaccines are being evaluated for safety and efficacy in healthy human subjects. A safety concern for any vaccine that contains genetic material, be it whole organism, live-attenuated, or gene-based, is the potential for integration into genomic DNA (gDNA). To address this concern, a preclinical pDNA persistence/integration study was conducted in rabbits to determine the level of pDNA in muscle 2, 28, and 64 days after intramuscular injection of DMRIE:DOPE-formulated pDNAs encoding Bacillus anthracis detoxified LF and PA proteins (VCL-AB01 vaccine). Total DNA was extracted from day 64 muscle tissue and fractionated by column agarose gel electrophoresis (CAGE). Plasmid copy number (PCN) in muscle 64 days after injection (geometric mean, 2808 PCN/microg of total DNA or 150,000 diploid genomes) was determined by quantitative polymerase chain reaction. Analysis of total DNA from five VCLAB01- injected rabbits revealed that two of five samples had no detectable PCN in the high molecular weight fraction after one round of CAGE, two samples had PCN under the lower limit of quantitation, and the remaining sample had 123 PCN/microg. All PCN in the latter sample cleared after an additional round of CAGE. It appears, therefore, that persisting PCN fractionate as low molecular weight material and are most likely not integrated into gDNA. Even if the worst-case assumption is made that the highest PCN found associated with gDNA represented covalently integrated pDNA inserts, the frequency of mutation would still be 500-fold lower than the autosomal spontaneous mutation rate.

Animals↗

A potential disruptive technology in vaccine development: gene-based vaccines and their application to infectious diseases.

Vaccine development requires an amalgamation of disparate disciplines and has unique economic and regulatory drivers. Non-viral gene-based delivery systems, such as formulated plasmid DNA, are new and potentially disruptive technologies capable of providing 'cheaper, simpler, and more convenient-to-use' vaccines. Typically and somewhat ironically, disruptive technologies have poorer product performance, at least in the near-term, compared with the existing conventional technologies. Because successful product development requires that the product's performance must meet or exceed the efficacy threshold for a desired application, the appropriate selection of the initial product applications for a disruptive technology is critical for its successful evolution. In this regard, the near-term successes of gene-based vaccines will likely be for protection against bacterial toxins and acute viral and bacterial infections. Recent breakthroughs, however, herald increasing rather than languishing performance improvements in the efficacy of gene-based vaccines. Whether gene-based vaccines ultimately succeed in eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria and tuberculosis, for which the conventional vaccine technologies have failed, remains to be determined. A success against any one of the persistent intracellular pathogens would be sufficient proof that gene-based vaccines represent a disruptive technology against which future vaccine technologies will be measured.

Communicable Disease Control↗

CpG oligodeoxynucleotide and Montanide ISA 51 adjuvant combination enhanced the protective efficacy of a subunit malaria vaccine.

Unmethylated CpG dinucleotide motifs present in bacterial genomes or synthetic oligodeoxynucleotides (ODNs) serve as strong immunostimulatory agents in mice, monkeys and humans. We determined the adjuvant effect of murine CpG ODN 1826 on the immunogenicity and protective efficacy of the Saccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP1(19)) of the murine malaria parasite Plasmodium yoelii. We found that in C57BL/6 mice, following sporozoite challenge, the degree of protective immunity against malaria induced by yMSP1(19) in a formulation of Montanide ISA 51 (ISA) plus CpG ODN 1826 was similar or superior to that conferred by yMSP1(19) emulsified in complete Freund's adjuvant (CFA/incomplete Freund's adjuvant). In total, among mice immunized with yMSP1(19), 22 of 32 (68.7%) with ISA plus CpG 1826, 0 of 4 (0%) with CFA/incomplete Freund's adjuvant, 0 of 4 (0%) with CpG 1826 mixed with ISA (no yMSP1(19)), and 0 of 11 (0%) with CpG 1826 alone were completely protected against development of erythrocytic stage infection after sporozoite challenge. The adjuvant effect of CpG ODN 1826 was manifested as both significantly improved complete protection from malaria (defined as the absence of detectable erythrocytic form parasites) (P = 0.007, chi square) and reduced parasite burden in infected mice. In vivo depletions of interleukin-12 and gamma interferon cytokines and CD4+ and CD8+ T cells in vaccinated mice had no significant effect on immunity. On the other hand, immunoglobulin G (IgG) isotype levels appeared to correlate with protection. Inclusion of CpG ODN 1826 in the yMSP1(19) plus ISA vaccine contributed towards the induction of higher levels of IgG2a and IgG2b (Th1 type) antibodies, suggesting that CpG ODN 1826 caused a shift towards a Th1 type of immune response that could be responsible for the higher degree of protective immunity. Our results indicate that this potent adjuvant formulation should be further evaluated for use in clinical trials of recombinant malarial vaccine candidates.

Adjuvants, Immunologic↗

Comparative analysis of the effects of packaging signal, transgene orientation, promoters, polyadenylation signals, and E3 region on growth properties of first-generation adenoviruses.

First-generation adenovectors have been developed for gene therapy and vaccine applications. The construction of these adenovectors has entailed the use of numerous types of expression cassettes. It has long been known that first-generation adenovectors can be rescued more easily and to higher titers with some transgenes than with others. This study has systematically shown that there can be marked differences in growth properties of recombinant adenovectors attributable to the use of promoters, the orientation of the transgene within the E1A/E1B-deleted region, and the inclusion of the E3 region. In addition, we had demonstrated the benefit of extending the packaging signal region to include elements V, VI, and VII. The effects of the complete packaging region were studied by plasmid competition studies between original and modified adenovectors. Similar competition studies between E3(+) and E3(-) adenovectors were performed and showed that the E3(+) vector had a growth advantage over its E3(-) counterpart. By making various changes, we have enhanced the growth capacity of our recombinant adenovector by more than 3-fold under serum-free and cell suspension growth conditions. Along with this enhanced growth, our adenovectors have maintained their genetic stability after 21 successive passages in cell culture. This increased robustness will be critical when adapting first-generation recombinant adenovectors to commercial production.

Adenoviridae↗

The effects of varying exposure to malaria transmission on development of antimalarial antibody responses in preschool children. XVI. Asembo Bay Cohort Project.

In areas of intense malaria transmission, malaria morbidity and mortality is highest in children 3-18 months old. Interventions that reduce malaria exposure early in life reduce morbidity but may also delay development of clinical immunity. We assessed the relationship between intensity of malaria exposure and development of antibody responses. Thirty-nine children were monitored monthly, from birth to > or =2.5 years old (1238 observations), and were divided into 3 exposure categories, on the basis of parasitemic episodes or entomological data. Children with low exposure during the first 2 years of life had higher subsequent levels of antibody to merozoite surface protein-1(19-kDa) (a marker of blood-stage responses) by months 24-35 (P<.05). This inverse relationship decreased as children aged. There was no consistent relationship between exposure early in life and subsequent levels of antibody to circumsporozoite protein (a marker of sporozoite-stage responses). These data suggest that, in areas of intense malaria transmission, during the first 3 years of life, interventions that either reduce the number of asexual parasitemic episodes or lower entomological exposure do not delay the development of antibody responses to blood-stage malarial antigens.

Animals↗

Development of an adenovirus-shedding assay for the detection of adenoviral vector-based vaccine and gene therapy products in clinical specimens.

Adenoviral vectors are used widely as gene therapy and vaccine delivery systems. An adenovirus-shedding assay may be performed in clinical trials to monitor the safety of the vector and to investigate the potential relation between clinical symptoms and shed vector virus. This report describes the development and statistical performance of the shedding assay. Live adenovirus was recovered from throat swab and urine samples spiked with E1-deleted adenovirus type 5 vector expressing HIV-1 gag [Ad5HIVgag], in the presence or absence of wild-type adenovirus (WT Ad5). Samples were cultured in 293 and A549 cells, and the DNA extracted from virus culture was tested by polymerase chain reaction (PCR) for sequence identity. The results showed that the frequency of Ad5HIVgag infectivity in 293 cells by cytopathic effect (CPE) or an immunofluorescence assay (IFA) was concentration-dependent (53% for 10(2), 94% for 10(4), and 100% for 10(6) viral particles). WT Ad5 virus did not interfere with Ad5HIVgag. PCR amplisets could specifically amplify target sequences in the background of nonspecific DNA matrices and could distinguish Ad5HIVgag from wild-type adenoviruses. This assay may be used for clinical trials using adenovirus vectors as vehicles for vaccines.

Adenoviridae↗

Aotus New World monkeys: model for studying malaria-induced anemia.

Falciparum malaria is a major cause of disease and death in African children and pregnant women, primarily due to severe anemia. We studied anemia in vaccinated Aotus monkeys during a second infection where the animals were considered to be semi-immune. Most animals had extremely low or undetectable levels of parasitemia; in some, anemia did not develop and reticulocytemia remained unchanged; in others, moderate to severe anemia developed with inappropriately low reticulocytemia indicating bone marrow dysfunction. Bone marrow rapidly responded after parasite clearance. The rapid drop in hematocrit despite extremely low to undetectable parasitemia indicated massive removal of uninfected red blood cells from the circulation that, in the presence of bone marrow dysfunction, led to severe anemia-the problem that occurs in African children. We demonstrate that Aotus monkeys are a nonhuman primate model to gain insight into the pathogenesis of severe anemia in African children.

Anemia↗

A DNA vaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 of Plasmodium falciparum induces antibody, interferon-gamma and cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory effects of granulocyte macrophage-colony stimulating factor.

We have constructed a DNA plasmid vaccine encoding the C-terminal 42-kDa region of the merozoite surface protein 1 (pMSP1(42)) from the 3D7 strain of Plasmodium falciparum (Pf3D7). This plasmid expressed recombinant MSP1(42) after in vitro transfection in mouse VM92 cells. Rhesus monkeys immunized with pMSP1(42) produced antibodies reactive with Pf3D7 infected erythrocytes by IFAT, and by ELISA against yeast produced MSP1(19) (yMSP1(19)). Immunization also induced antigen specific T cell responses as measured by interferon-gamma production, and by classical CTL chromium release assays. In addition, immunization with pMSP1(42) primed animals for an enhanced antibody response to a subsequent boost with the recombinant yMSP1(19). We also evaluated Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as an adjuvant for pMSP1(42.) We tested both rhesus GM-CSF expressed from a DNA plasmid, and E. coli produced recombinant human GM-CSF. Plasmids encoding rhesus GM-CSF (prhGM-CSF) and human GM-CSF (phuGM-CSF) were constructed; these plasmids expressed bio-active recombinant GMCSF. Co-immunization with a mixture of prhGM-CSF and pMSP1(42) induced higher specific antibody responses after the first dose of plasmid, but after three doses of DNA monkeys immunized with or without prhGM-CSF had the same final antibody titers and T cell responses. In comparison, rhuGM-CSF protein did not lead to accelerated antibody production after the first DNA dose. However, antibody titers were maintained at a slightly higher level in monkeys receiving GM-CSF protein, and they had a higher response to boosting with recombinant MSP1(19). The GM-CSF plasmid or protein appears to be less potent as an adjuvant in rhesus monkeys than each is in mice, and more work is needed to determine if GM-CSF can be a useful adjuvant in DNA vaccination of primates.

Adjuvants, Immunologic↗

Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity.

Recent studies of human immunodeficiency virus type 1 (HIV-1) infection in humans and of simian immunodeficiency virus (SIV) in rhesus monkeys have shown that resolution of the acute viral infection and control of the subsequent persistent infection are mediated by the antiviral cellular immune response. We comparatively assessed several vaccine vector delivery systems-three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector-expressing the SIV gag protein for their ability to elicit such immune responses in monkeys. The vaccines were tested either as a single modality or in combined modality regimens. Here we show that the most effective responses were elicited by a replication-incompetent Ad5 vector, used either alone or as a booster inoculation after priming with a DNA vector. After challenge with a pathogenic HIV-SIV hybrid virus (SHIV), the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection. The replication-defective adenovirus is a promising vaccine vector for development of an HIV-1 vaccine.

AIDS Vaccines↗

Improved immunogenicity and efficacy of the recombinant 19-kilodalton merozoite surface protein 1 by the addition of oligodeoxynucleotide and aluminum hydroxide gel in a murine malaria vaccine model.

Vaccination of mice with yeast-secreted Plasmodium yoelii-derived 19-kilodalton merozoite surface protein 1 (yMSP1(19)) has been shown to afford protection from challenge with a lethal strain of P. yoelii. Sterile immunity can be achieved when MSP1(19) is emulsified in Freund adjuvant but not when it is adsorbed to aluminum hydroxide gel (alum). Because complete Freund adjuvant is not an acceptable adjuvant for use in humans, alternative adjuvants must be identified for formulating MSP1(19) as a vaccine for use in humans. To determine whether oligodeoxynucleotides with CpG motifs (ODN), reported to be a powerful new class of adjuvants, could enhance the immunogenicity of yMSP1(19), C57BL/6 mice were vaccinated either with yMSP1(19) formulated with Freund adjuvant, with alum, or with ODN plus alum and challenged intravenously with P. yoelii 17XL asexual blood-stage parasites. Adsorption of immunogen and adjuvant to alum was optimized by adjusting buffer (phosphate versus acetate) and pH. We found that the adjuvant combination of ODN plus alum with yMSP1(19), injected intraperitoneally (i.p.), increased immunoglobulin G (IgG) yMSP1(19)-specific antibody production 12-fold over Freund adjuvant given i.p., 3-fold over Freund adjuvant given subcutaneously (s.c.), 300-fold over alum given i.p., and 48-fold over alum given s.c. The predominant antibody isotype in the group receiving alum-ODN-yMSP1(19) was IgG1. Increased antibody levels correlated to protection from a challenge with P. yoelii 17XL. Supernatant cytokine levels of gamma interferon in yMSP1(19)-stimulated splenocytes were dramatically elevated in the alum-ODN-yMSP1(19) group. Interleukin-10 (IL-10) levels were also elevated; however, no IL-5 was detected. The cytokine profile, as well as the predominant IgG1 antibody isotype, suggests the protective immune response was a mixed Th1/Th2 response.

Adjuvants, Immunologic↗

Regulation of antigen-specific immunoglobulin G subclasses in response to conserved and polymorphic Plasmodium falciparum antigens in an in vitro model.

Cytophilic antibodies (Abs) play a critical role in protection against Plasmodium falciparum blood stages, yet little is known about the parameters regulating production of these Abs. We used an in vitro culture system to study the subclass distribution of antigen (Ag)-specific immunoglobulin G (IgG) produced by peripheral blood mononuclear cells (PBMCs) from individuals exposed to P. falciparum or unexposed individuals. PBMCs, cultivated with or without cytokines and exogenous CD40/CD40L signals, were stimulated with a crude parasite extract, recombinant vaccine candidates derived from conserved Ags (19-kDa C terminus of merozoite surface protein 1 [MSP1(19)], R23, and PfEB200), or recombinant Ags derived from the polymorphic Ags MSP1 block 2 and MSP2. No P. falciparum-specific Ab production was detected in PBMCs from unexposed individuals. PBMCs from donors exposed frequently to P. falciparum infections produced multiple IgG subclasses when they were stimulated with the parasite extract but usually only one IgG subclass when they were stimulated with a recombinant Ag. Optimal Ab production required addition of interleukin-2 (IL-2) and IL-10 for all antigenic preparations. The IgG subclass distribution was both donor and Ag dependent and was only minimally influenced by the exogenous cytokine environment. In vitro IgG production and subclass distribution correlated with plasma Abs to some Ags (MSP1(19), R23, and MSP2) but not others (PfEB200 and the three MSP1 block 2-derived Ags). Data presented here suggest that intrinsic properties of the protein Ag itself play a major role in determining the subclass of the Ab response, which has important implications for rational design of vaccine delivery.

Adult↗

Short report: differential evolution of immunoglobulin G1/G3 antibody responses to Plasmodium falciparum MSP1(19) over time in malaria-immune adult Senegalese patients.

This study examined the evolution of immunoglobulin (Ig) G1 and IgG3 antibodies against the asexual stage Plasmodium falciparum protein, MSP1(19), before and after a heavy malaria transmission period in clinically immune Senegalese subjects living under different epidemiological conditions. Plasma was tested for antibodies to a yeast-produced, recombinant PfMSP1(19) antigen (the Q-KNG allelic variant) that has previously been demonstrated to react with IgG1, IgG3, or both in the majority of these people. Anti-P. falciparum antibodies of the IgG1 and IgG3 subclasses, previously reported to be associated with protection, were shown to evolve independently one from another after the transmission period in both settings. These results suggest differential regulation of MSP1(19)-specific IgG1 and IgG3. The precise role of these antibody isotypes in maintaining malaria immunity remains to be determined.

Adult↗