Anders Fomsgaard

Gene gun DNA vaccination with Rev-independent synthetic HIV-1 gp160 envelope gene using mammalian codons.

DNA immunization with HIV envelope plasmids induce only moderate levels of specific antibodies which may in part be due to limitations in expression influenced by a species-specific and biased HIV codon usage. We compared antibody levels, Th1/Th2 type and CTL responses induced by synthetic genes encoding membrane bound gp160 versus secreted gp120 using optimized codons and the efficient gene gun immunization method. The in vitro expression of syn.gp160 as gp120 + gp41 was Rev independent and much higher than a classical wt.gp160 plasmid. Mice immunized with syn.gp160 and wt.gp160 generated low and inconsistent ELISA antibody titres whereas the secreted gp120 consistently induced faster seroconversion and higher antibody titres. Due to a higher C + G content the numbers of putative CpG immune (Th1) stimulatory motifs were highest in the synthetic gp160 gene. However, both synthetic genes induced an equally strong and more pronounced Th2 response with higher IgG1/IgG2a and IFNgamma/IL-4 ratios than the wt.gp160 gene. As for induction of CTL, synthetic genes induced a somewhat earlier response but did not offer any advantage over wild type genes at a later time point. Thus, optimizing codon usage has the advantage of rendering the structural HIV genes Rev independent. For induction of antibodies the level of expression, while important, seems less critical than optimal contact with antigen presenting cells at locations reached by the secreted gp120 protein. A proposed Th1 adjuvant effect of the higher numbers of CpG motifs in the synthetic genes was not seen using gene gun immunization which may be due to the low amount of DNA used.

First introduction of highly pathogenic H5N1 avian influenza A viruses in wild and domestic birds in Denmark, Northern Europe

BackgroundSince 2005 highly pathogenic (HP) avian influenza A H5N1 viruses have spread from Asia to Africa and Europe infecting poultry, humans and wild birds. HP H5N1 virus was isolated in Denmark for the first time in March 2006. A total of 44 wild birds were found positive for the HP H5N1 infection. In addition, one case was reported in a backyard poultry flock.ResultsFull-genome characterisation of nine isolates revealed that the Danish H5N1 viruses were highly similar to German H5N1 isolates in all genes from the same time period. The haemagglutinin gene grouped phylogenetically in H5 clade 2 subclade 2 and closest relatives besides the German isolates were isolates from Croatia in 2005, Nigeria and Niger in 2006 and isolates from Astrakhan in Russia 2006. The German and Danish isolates shared unique substitutions in the NA, PB1 and NS2 proteins.ConclusionThe first case of HP H5N1 infection of wild and domestic birds in Denmark was experienced in March 2006. This is the first full genome characterisation of HP H5N1 avian influenza A virus in the Nordic countries. The Danish viruses from this time period have their origin from the wild bird strains from Qinghai in 2005. These viruses may have been introduced to the Northern Europe through unusual migration due to the cold weather in Eastern Europe at that time.

Hepatitis C Virus Subtyping by a Core-Envelope 1-Based Reverse Transcriptase PCR Assay with Sequencing and Its Use in Determining Subtype Distribution among Danish Patients

ABSTRACT A reverse transcriptase PCR (RT-PCR) assay using conserved primers deduced from the core-envelope 1 (C-E1) region of the hepatitis C virus (HCV) genome was developed for subtyping purposes. The sensitivity and specificity of this assay tested against two HCV reference panels containing genotype 1 through 5 subtypes were similar to those of an RT-PCR assay from the 5′-untranslated region (5′-UTR). The sensitivity of the RT-PCR typing assay in the more variable C-E1 region was, however, lower than that of the RT-PCR in the highly conserved 5′-UTR when testing multiple clinical samples. Thus, 71 (88%) of 81 consecutive samples from hospitalized Danish patients positive for HCV antibodies and RNA (5′-UTR) were positive also in the C-E1 RT-PCR assay. Phylogenetic analysis of the E1 sequences obtained by direct sequencing of HCV from two reference panels and 71 Danish patients allowed us to readily distinguish the subtypes. In contrast, phylogenetic analysis of their corresponding 5′-UTR sequences was able to predict only major genotypes. Three different genotypes and four subtypes were identified among Danish samples: 1a (43%), 1b (11%), 2b (6%), and 3a (39%). An isolate from a Somalian refugee was identified as a new HCV type related to Somalian isolates described as subtype 3h. The most common genotype in Denmark is genotype 1 (53%), which is the most difficult to treat. However, Denmark had the highest prevalence in Europe of subtype 3a, which responds more favorably to treatment. The described C-E1 RT-PCR with sequencing is suggested as an easy routine assay for definitive genotyping and subtyping of HCV.

Low level of regulatory T cells and maintenance of balance between regulatory T cells and TH17 cells in HIV-1-infected elite controllers.

Background:A subgroup of HIV-1-infected individuals, elite controllers, have spontaneous viral control and offer an exceptional opportunity to study virological and immunolocigal factors of possible involvement in control of HIV-1 infection. Methods:The frequencies of Tregs and TH17 cells was evaluated and correlated to markers of disease progression in peripheral blood mononuclear cells from 3 different groups of individuals infected with HIV-1: treatment-naive viremic individuals, individuals on successful highly active antiretroviral therapy, and elite controllers. In addition, a group of HIV-1-negative individuals were included. Results:We demonstrate that elite controllers have lower levels of Tregs compared with HIV-1-infected viremic individuals, but that the low Treg level does not differ between individuals with HIV-1 control, whether natural or therapy induced. We also show that T-cell activation and proliferation both correlate to the level of Tregs. Finally, the TH17/Treg ratio was similar in Elite Controllers and uninfected controls, whereas in viremic and treated HIV-1-infected individuals, the TH17/Treg ratio was lower compared with uninfected controls. Conclusions:We show that one feature of spontaneous HIV-1 control is a maintained balance between regulatory T cells and TH17 cells.

Induction of novel CD8+ T-cell responses during chronic untreated HIV-1 infection by immunization with subdominant cytotoxic T-lymphocyte epitopes.

Objective:To investigate the potential to induce additional cytotoxic T-lymphocyte (CTL) immunity during chronic HIV-1 infection. Design:We selected infrequently targeted or subdominant but conserved HLA-A*0201-binding epitopes in Gag, Pol, Env, Vpu and Vif. These relatively immune silent epitopes were modified as anchor-optimized peptides to improve immunogenicity and delivered on autologous monocyte-derived dendritic cells (MDDCs). Methods:Twelve treatment-naïve HLA-A*0201 HIV-1-infected Danish individuals received 1 × 107 MDDCs subcutaneously (s.c.) (weeks 0, 2, 4 and 8), pulsed with seven CD8+ T-cell epitopes and three CD4+ T-cell epitopes. Epitope-specific responses were evaluated by intracellular cytokine staining for interferon-γ, tumor necrosis factor α and interleukin-2 and/or pentamer labeling 3 weeks prior to, 10 weeks after and 32 weeks after the first immunization. Results:Previously undetected T-cell responses specific for one or more epitopes were induced in all 12 individuals. Half of the participants had sustained CD4+ T-cell responses 32 weeks after immunization. No severe adverse effects were observed. No overall or sustained change in viral load or CD4+ T-cell counts was observed. Conclusion:These data show that it is possible to generate new T-cell responses in treatment-naive HIV-1-infected individuals despite high viral loads, and thereby redirect immunity to target new multiple and rationally selected subdominant CTL epitopes. Further optimization could lead to stronger and more durable cellular responses to selected epitopes with the potential to control viral replication and prevent disease in HIV-1-infected individuals.

Improved Humoral and Cellular Immune Responses Against the gp120 V3 Loop of HIV-1 Following Genetic Immunization with a Chimeric DNA Vaccine Encoding the V3 Inserted into the Hepatitis B Surface Antigen

The gp120‐derived V3 loop of HIV‐1 is involved in co‐receptor interaction, it guides cell tropism, and contains an epitope for antibody neutralization. Thus, HIV‐1 V3 is an attractive vaccine candidate. The V3 of the MN strain (MN V3) contains both B‐ and T‐cell epitopes, including a known mouse H‐2d‐restricted cytotoxic T lymphocyte (CTL) epitope. In an attempt to improve the immunogenicity of V3 in DNA vaccines, a plasmid expressing MN V3 as a fusion protein with the highly immunogenic middle (pre‐S2 + S) surface antigen of hepatitis B virus (HBsAg) was constructed. Epidermal inoculation by gene gun was used for genetic immunization in a mouse model. Antibody and CTL responses to MN V3 and HBsAg were measured and compared with the immune responses obtained after vaccination with plasmids encoding the complete HIV‐1 MN gp160 and HBsAg (pre‐S2 + S), respectively. DNA vaccination with the HIV MN gp160 envelope plasmid induced a slow and low titred anti‐MN V3 antibody response at 12 weeks post‐inoculation (p.i.) and a late appearing (7 weeks), weak and variable CTL response. In contrast, DNA vaccination with the HBsAg‐encoding plasmid induced a rapid and high titred anti‐HBsAg antibody response and a uniform strong anti‐HBs CTL response already 1 week p.i. in all mice. DNA vaccination with the chimeric MN V3/HBsAg plasmid elicited humoral responses against both viruses within 3–6 weeks which peaked at 6–12 weeks and remained stable for at least 25 weeks. In addition, specific CTL responses were induced in all mice against both MN V3 and HBsAg already within the first 3 weeks, lasting at least 11 weeks. Thus, HBsAg acts as a ‘genetic vaccine adjuvant’ augmenting and accelerating the cellular and humoral immune response against the inserted MN V3 loop. Such chimeric HIV–HBsAg plasmid constructs may be useful in DNA immunizations as a ‘carrier’ of protein regions or minimal epitopes which are less exposed or poorly immunogenic.

HIV-1 DNA vaccines.

HIV-1 was among the original DNA vaccine targets and HIV DNA vaccines are now in human trials. Lack of strong correlates of protective immunity makes vaccine design difficult; however, DNA vaccines have the potential to be an ideal vaccine and therapeutic approach against HIV-1. DNA vaccines induce conformational-dependent antibodies, mimic live vaccines but without the pathogenic potential, and can easily be made polyvalent. Genes which encode important CTL and antibody epitopes can be included while those that confer pathogenicity, virulence, antibody enhancement or represent non-conserved epitopes can be excluded. In our hands pre-treatment of muscles with bupivacaine or cardiotoxin did not offer any advantage over no muscle pre-treatment or gene gun inoculation of skin although gene gun immunization seem to favour a Th2 type response. As DNA vaccine candidates we have compared vaccines encoding native HIV MN gp160 with Rev-independent synthetic genes encoding MNgp160 and MNgp120 using mammalian high expression codons. In these experiments the gene encoding secreted gp120 gave highest antibody neutralizing titers. High and fast antibody responses could also be obtained by transferring the HIV-1 MN V3 loop to the secreted HBsAg as a fusion gene vaccine. Thus, in the case of HIV-1 MN genes encoding secreted surface glycoproteins may be preferred instead of membrane bound envelopes. CTL responses were induced in all cases. However, in order to meet the high diversity of HIV and HLA types our approach is to include many CTL epitopes in a multivalent minigene vaccine. We found that gene gun DNA vaccination with minimal epitopes could induce specific CTL. Flanking sequences influenced the CTL response but was not needed. DNA vaccines encoding known and computer predicted CTL epitopes are now being developed.

Multiple human papilloma virus types in cervical infections: competition or synergy?

Coinfection with multiple human papilloma virus (HPV) types is common in cervical HPV infection. To evaluate if infections with different HPV types occur independently, we examined 3558 women above 15 years of age suspected of cervical HPV infection. Among them, 1842 (52%) women were HPV negative and 1716 (48%) were HPV positive as analysed by a PCR-based commercial microarray assay for mucosal types. Of the HPV-positive samples, 824 (48%) had single infections, while 892 (52%) had multiple infections. Observed numbers of concurrent HPV types differed from expected numbers under the assumption of independence between infections by the various HPV types. Significant positive associations were observed for 16 pairs of HPV types in statistical analysis accounting for mass significance. Significant negative associations were also found, i.e. women with HPV-16 infection had 0.4 times the odds of having HPV-51 compared with women not infected with HPV-16. HPV-16 was the only type with odds ratios <1 for all pairwise combinations. While our findings of statistically significant coexistence do not prove biological dependence among HPV types, they do suggest that infections with some HPV types may depend on the existence of certain other HPV types. Any interaction between coexisting HPV types could either decrease or increase the efficacy of current HPV vaccines that offer mainly type-specific protection, depending on whether the types vaccinated against compete with other HPV types or not.

High frequency of multiple HPV types in cervical specimens from Danish women

Genital human papillomavirus infection (HPV) is common and usually harmless. However, chronic cervical infection with high‐risk HPV types can cause cell changes that may eventually lead to cancer. To determine the frequency of individual HPV types among mixed infections, we examined the type distribution among cervical specimens from more than 1000 Danish women. We also examined the HPV type distribution and the frequency of single and multiple HPV types for specimens from 113 women who underwent conization and were diagnosed with cervical intraepithelial neoplasia grade II or worse (CIN2+). Using microarray technology, we found that 49% of the HPV‐positive patients were infected with multiple HPV types. Among the CIN2+ diagnosed women, this frequency was 41%. The most frequently found high‐risk HPV type was HPV‐16, which was found in 25% of the HPV‐positive cervical specimens. Among the HPV positive CIN2+ diagnosed women, 48% were HPV‐16 positive. Women younger than 30 years of age had a higher frequency of multiple infections (61%) than women older than 30 years (39%). We conclude that cervical infection with multiple HPV types is common among women in all age groups and among women with or without the diagnosis of CIN2+.

Multiple human papilloma virus types in cervical infections: competition or synergy?: HPV COMPETITION

Mejlhede N, Pedersen BV, Frisch M, Fomsgaard A. Multiple human papilloma virus types in cervical infections: competition or synergy? APMIS 2010; 118: 346–52.