A case of paramyotonia congenita in pregnancy.

Paramyotonia congenita is a rare autosomal dominant non-dystrophic myopathy caused by mutations in the SNC4A gene, which encodes for the voltage-gated sodium channel in skeletal muscle. Symptom onset is typically during early childhood and is characterised by myotonia followed by flaccid paralysis or weakness, usually exacerbated by repeated muscle contractions or cold temperatures. Pregnancy has been reported to increase symptoms of myotonia; however, there is limited information in the literature regarding the possible effects of paramyotonia congenita on pregnancy and labour. We present a successful case of a 20-year-old primigravida with confirmed paramyotonia congenita and review the literature regarding paramyotonia congenita during pregnancy.

HIV/AIDS vaccines: 2007.

Development of an HIV/AIDS vaccine has been slow because classical approaches to vaccine development have not yielded a vaccine. Encouragingly, new approaches using recombinant viral vectors, DNA vaccines, and combinations of different vectors in heterologous prime/boost regimens are yielding vaccines capable of controlling virulent immunodeficiency virus challenges in non-human primate models. These new vaccines elicit T cells capable of recognizing and killing virus-infected cells. Brief synopses are given for six vaccines currently advancing in human trials.

Excellent safety and tolerability of the human immunodeficiency virus type 1 pGA2/JS2 plasmid DNA priming vector vaccine in HIV type 1 uninfected adults.

A vaccine consisting of DNA priming followed by recombinant modified vaccinia Ankara (rMVA) boosting has achieved long-term control of a pathogenic challenge with a chimera of simian and human immunodeficiency viruses (SHIV-89.6P) in rhesus macaques. Based on these results, clade B HIV-1 DNA and rMVA immunogens have been developed for trials in humans. We conducted a first-time in humans phase I safety trial using the pGA2/JS2 (JS2) HIV-1 DNA priming vector expressing Gag, Pol, Env, Tat, Rev, and Vpu. Thirty HIV-uninfected adults were vaccinated with 0.3 or 3 mg of JS2 DNA, or a saline placebo, by intramuscular injection at months 0 and 2. Both doses of DNA were safe and well-tolerated with no differences between the control, 0.3 mg, or 3 mg groups (n = 6, 12, and 12, respectively) through 12 months of postvaccination follow- up. A chromium-release assay using fresh peripheral blood mononuclear cells (PBMCs) and a validated IFN-gamma ELISpot assay with frozen PBMCs failed to detect CD4(+) or CD8(+) HIV-1-specific T cell responses. HIV-specific neutralizing antibodies were also not detected. The vaccine is being further developed as a priming vector for a combined DNA plus rMVA prime/boost HIV vaccination regimen.

DNA prime/protein boost vaccine strategy in neonatal macaques against simian human immunodeficiency virus.

Newborn macaques were vaccinated against a chimeric simian human immunodeficiency (SHIV) virus, SHIV-vpu+, by DNA priming and boosting with homologous HIV-1 gp160. Following SHIV-vpu+ challenge, containment of infection was observed in 4 of 15 animals given DNA priming/protein boost vaccination and in three of four animals given gp160 boosts only. Rechallenge with homologous virus of six animals that contained the first challenge virus resulted in rapid viral clearance or low viral loads. Upon additional rechallenge with heterologous, pathogenic SHIV89.6P, four of these six animals maintained normal CD4+ T-cell counts with no or limited SHIV89.6P infection. Our data suggest that humoral and cellular immune mechanisms may have contributed to the containment of SHIV89.6P; however, viral interference with SHIV-vpu+ could also have played a role. Our results indicate that immunogenicity and efficacy of candidate AIDS vaccines are not affected when vaccination is initiated during infancy as compared with later in life.

Isogamous, hermaphroditic inheritance of mitochondrion-encoded resistance to Qo inhibitor fungicides in Blumeria graminis f. sp. tritici.

A mutation of glycine to alanine at position 143 in the mitochondrial cytochrome b amino acid sequence of Blumeria graminis f. sp. tritici cosegregated with the QoI-resistant phenotype in a ratio of 1:1 in a cross between a sensitive and a resistant isolate. This mutation was used as a mitochondrial marker to determine whether mitochondrial inheritance in B. graminis was anisogamous, as in heterothallic Neurospora sp., or isogamous and hermaphroditic, as in Aspergillus nidulans. Segregation of mitochondrial genotypes in B. graminis f. sp. tritici was consistent with inheritance of mitochondria being hermaphroditic and isogamous, in that all ascospores from an individual cleistothecium had the same mitochondrial genotype and that either parent could act as the maternal parent of a cleistothecium. Within each cleistothecium, nuclear segregation occurred independently of mitochondrial inheritance, as shown by segregation of resistance to the fungicide triadimenol and by segregation of avirulences to the wheat cultivars Galahad (Pm2), Armada (Pm4b), and Holger (Pm6).

Heterologous neutralizing antibody induction in a simian-human immunodeficiency virus primate model: lack of original antigenic sin.

According to the principle of original antigenic sin, neutralizing antibodies (NAbs) initially directed against a single virus strain compromise the immune system's ability to subsequently mount adequate responses against antigenically divergent virus strains. In this study, rhesus macaques, after vaccination and breakthrough infection with homologous simian-human immunodeficiency virus (SHIV), developed strong SHIV-IIIB strain-directed NAb responses that were mostly V3 loop specific. After superinfection with heterologous SHIV89.6P, all macaques developed high-titer SHIV89.6P-specific NAbs without significant boosting of SHIV-IIIB-specific NAbs. These results indicate that prior B cell responses against a single immunodeficiency virus strain do not preclude the later development of NAbs against a divergent strain of the same virus.

C3d enhancement of neutralizing antibodies to measles hemagglutinin.

Measles remains a major cause of worldwide infant mortality despite the use of current live attenuated vaccines. New approaches to measles virus (MV) vaccine development are critical to interrupt the spread of MV. In this study, we report the results using a DNA vaccine expressing a fusion of the measles hemagglutinin (H) protein and the complement component, C3d, to enhance the titers of neutralizing antibody. Plasmids were generated that expressed a secreted (s) form of H and the same form fused to three tandem copies of the murine homologue of C3d (sH-3C3d). Analysis of titers of the antibody raised in vaccinated mice indicated that immunizations with the DNA expressing sH-3C3d had higher titers of anti-H antibodies compared to serum from mice vaccinated with DNA expressing sH only. In addition, sH-3C3d elicited higher neutralizing antibody titers that inhibited MV induced plaque formation.

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.

Enhanced avidity maturation of antibody to human immunodeficiency virus envelope: DNA vaccination with gp120-C3d fusion proteins.

DNA vaccination can elicit both humoral and cellular immune responses and can confer protection against several pathogens. However, DNA vaccines expressing the envelope (Env) protein of human immunodeficiency virus (HIV) have been relatively ineffective at generating high titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, we report that fusion of Env and the complement component, C3d, in a DNA vaccine, enhances the titers of antibody to Env. Plasmids were generated that expressed a secreted form of Env (sgp120) from three isolates of HIV and these same forms fused to three tandem copies of the murine homologue of C3d (sgp120-3C3d). Analyses of titers and avidity maturation of the raised antibody indicated that immunizations with each of the sgp120-3C3d-expressing DNAs accelerated both the onset and the avidity maturation of antibody to Env.

Apoptosis-mediated enhancement of DNA-raised immune responses by mutant caspases.

Apoptotic bodies can be used to target delivery of DNA-expressed immunogens into professional antigen-presenting cells (APCs). Here we show that antigen-laden apoptotic bodies created by vectors co-expressing influenza virus hemagglutinin (HA) or nucleoprotein (NP) genes and mutant caspase genes markedly increased T-cell responses. Both CD8 and CD4 T-cell responses were affected. The adjuvant activity was restricted to partially inactivated caspases that allowed immunogen expression before the generation of apoptotic bodies. Active-site mutants of murine caspase 2 and an autocatalytic chimera of murine caspase 2 prodomain and human caspase 3 induced apoptosis that did not interfere with immunogen expression. The adjuvant activity also enhanced B-cell responses, but to a lesser extent than T-cell responses. The large increases in T-cell responses represent one of the strongest effects to date of a DNA adjuvant on cellular immunity.

Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques.

The ability to generate antibodies that cross-neutralize diverse primary isolates is an important goal for human immunodeficiency virus type 1 (HIV-1) vaccine development. Most of the candidate HIV-1 vaccines tested in humans and nonhuman primates have failed in this regard. Past efforts have focused almost entirely on the envelope glycoproteins of a small number of T-cell line-adapted strains of the virus as immunogens. Here we assessed the immunogenicity of noninfectious virus-like particles (VLP) consisting of Gag, Pro (protease), and Env from R5 primary isolate HIV-1(Bx08). Immunogens were delivered to rhesus macaques in the form of either purified VLP, recombinant DNA and canarypox (ALVAC) vectors engineered to express VLP, or a combination of these products. Seroconversion to Gag and Pro was detected in all of the immunized animals. Antibodies that could neutralize HIV-1(Bx08) were detected in animals that received (i) coinoculations with DNA(Bx08) and VLP(Bx08), (ii) DNA(Bx08) followed by ALVAC(Bx08) boosting, and (iii) VLP(Bx08) alone. The neutralizing antibodies were highly strain specific despite the fact that they did not appear to be directed to linear epitopes in the V3 loop. Virus-specific cellular immune responses also were generated, as judged by the presence of Gag-specific gamma interferon (IFN-gamma)-producing cells. These cellular immune responses required the inclusion of DNA(Bx08) in the immunization modality, since few or no IFN-gamma-producing cells were detected in animals that received either VLP(Bx08) or ALVAC(Bx08) alone. The results demonstrate the feasibility of generating neutralizing antibodies and cellular immune responses that target an R5 primary HIV-1 isolate by vaccination in primates.

Simian immunodeficiency virus (SIV) infection of a rhesus macaque induces SIV-specific CD8(+) T cells with a defect in effector function that is reversible on extended interleukin-2 incubation.

A vigorous expansion of antigen-specific CD8(+) T cells lacking apparent effector function was observed in a rhesus macaque acutely infected with the simian immunodeficiency virus (SIV) strain SIVmac239. Antigen-specific CD8(+) T cells were identified using antigenic-peptide class I major histocompatibility complex tetramers. As many as 8.3% of CD8(+) cells recognized the Mamu-A*01-associated SIV epitope Gag(181-189) (CTPYDINQM); however, these cells demonstrated no effector function when presented with peptide-incubated targets, as measured by intracellular cytokine staining for gamma interferon (IFN-gamma), interleukin-2 (IL-2) production, or direct cellular lysis. Similar results were observed with three other SIV peptide antigens. Nonresponsiveness did not correlate with apoptosis of the CD8(+) cells, nor were cells from this macaque impaired in their ability to present peptide antigens. Associated with the nonresponsive state was a lack of IL-2 production and decreased IL-2 receptor expression. Exogenous IL-2 treatment for 1 week in the absence of antigenic stimulation restored antigen-specific responses and the quantitative correlation between tetramer recognition and antigen-responsive IFN-gamma secretion. This case report suggests a regulatory mechanism that may impede the effector function of antigen-specific T cells during acute infection with SIV or human immunodeficiency virus in some cases. This mechanism may participate in the failure of the immune system to limit infection.

Th1 genetic adjuvants modulate immune responses in neonates.

In these studies, we address the ability of DNA encoding Th1 cytokines to bias the isotype of antibody raised by neonatal or adult immunization with an influenza hemagglutinin expressing DNA (HA-DNA). Neonatal mice coimmunized with HA-DNA and either IL-12 or IFN-gamma-expressing DNA developed IgG2a-biased immune responses, regardless of inoculation method. In contrast, the Th1 genetic adjuvants had no effect on IgG subtype patterns in adults. In neonatal mice, the Th1 genetic adjuvants also shifted the pattern of lymphokine production by recall splenocytes from a mixed response of IFN-gamma and IL-5 to exclusively IFN-gamma. In adults, despite the failure to change the isotype pattern of the antibody response, a shift towards IFN-gamma production also occurred for recall splenocytes following coimmunzation with IL-12. Thus, coinoculation of Th1 genetic adjuvants had greater effects on the nature of the immune response in the neonate than in adults.

Nucleic acid immunizations.

This unit provides protocols for two broadly used methods of DNA immunization: saline injections and gene gun deliveries of DNA. Saline injections deliver DNA into extracellular spaces; gene gun deliveries bombard DNA directly into cells. Support protocols present methods for preparation of DNA-coated gold beads, creation of cartridges containing these beads, and optimization of gene gun parameters. Issues relating to plasmid vectors, DNA preparation and doses, and immunization regimens are also discussed. Expression library immunizations, genetic and conventional adjuvants, alternative boosts, neonatal immunizations, and approaches to mucosal delivery of DNA are covered in the commentary.

DNA vaccines for influenza virus: differential effects of maternal antibody on immune responses to hemagglutinin and nucleoprotein.

Maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature; however, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. We tested the ability of intramuscular and gene gun immunization with DNA expressing influenza virus hemagglutinin (HA) and nucleoprotein (NP) to raise protective humoral and cellular responses in the presence or absence of maternal antibody. Neonatal mice born to influenza virus-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD8(+) T-cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses but did affect humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell.

Successful DNA immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles.

Measles remains a principal cause of worldwide mortality, in part because young infants cannot be immunized effectively. Development of new vaccines has been hindered by previous experience with a formalin-inactivated vaccine that predisposed to a severe form of disease (atypical measles). Here we have developed and tested potential DNA vaccines for immunogenicity, efficacy and safety in a rhesus macaque model of measles. DNA protected from challenge with wild-type measles virus. Protection correlated with levels of neutralizing antibody and not with cytotoxic T lymphocyte activity. There was no evidence in any group, including those receiving hemagglutinin-encoding DNA alone, of 'priming' for atypical measles.

Basic mechanisms of DNA-raised antibody responses to intramuscular and gene gun immunizations.

DNA-raised antibody (Ab) responses have been compared for the dependence on CD4+ and CD8+ cells, the longevity of functional antigen (Ag) expression, and the nature of the Ag-presenting cell after intramuscular (i.m.) and gene gun inoculations. A plasmid expressing the hemagglutinin (HA) glycoprotein of influenza virus was used for immunizations of BALB/c mice. Intramuscular and gene gun-raised Abs had similar dependencies on CD4+ and CD8+ cells but different temporal patterns of functional Ag expression. The two methods of DNA immunization also appeared to have different frequencies or types of Ag-presenting cells in the draining lymph nodes and spleen. For both methods of DNA delivery, Ab was independent of CD8+ cells but dependent on CD4+ cells. The CD4 dependence occurred at priming but not booster immunizations and resulted in a 1-month delay in the Ab response. Temporal T-cell transfers from TCR+/+ mice into immunized TCR-/- mice revealed the persistence of DNA-expressed Ag for up to 1 month after both i.m. and gene gun inoculations. For gene gun, but not i.m. immunizations, approximately 90% of the functional Ag expression was lost by 1 week, consistent with the sloughing of the epidermal target site. Despite similar titers of raised Ab, Ag-presenting dendritic cells could be detected in the draining lymph nodes and spleen of gene gun- but not i.m. DNA-immunized mice. In the gene gun-immunized mice, Ag-presenting dendritic cells appeared in the draining lymph nodes before the spleen.

C3d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge.

The ability of the C3d component of complement to enhance antibody responses and protective immunity to influenza virus challenges was evaluated using a DNA vaccine encoding a C3d fusion of the hemagglutinin (HA) from influenza virus. Plasmids were generated that encoded a transmembrane HA (tmHA), a secreted form of HA (sHA), or a sHA fused to three tandem copies of the murine homologue of the C3d (sHA-3C3d). Analysis of the titers, avidity maturation, and hemagglutinin-inhibition activity of raised antibody revealed that immunizations with sHA-3C3d DNA accelerated both the avidity maturation of antibody to HA and the appearance of hemagglutinin-inhibition activity. These accelerated antibody responses correlated to a more rapid appearance of protective immunity. They also correlated to complete protection from live virus challenge by a single vaccination at a dose ten times lower than the protective dose for non-C3d forms of HA.

DNA priming and recombinant pox virus boosters for an AIDS vaccine.

Eight different protocols for immunization have been compared for the ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.