Marie Borggren

Increased Sensitivity to Broadly Neutralizing Antibodies of End-Stage Disease R5 HIV-1 Correlates with Evolution in Env Glycosylation and Charge.

Background Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset. Principal Findings HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4+ T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope. Conclusions Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge.

Rational design of HIV vaccines and microbicides: report of the EUROPRISE network annual conference 2010

Novel, exciting intervention strategies to prevent infection with HIV have been tested in the past year, and the field is rapidly evolving. EUROPRISE is a network of excellence sponsored by the European Commission and concerned with a wide range of activities including integrated developmental research on HIV vaccines and microbicides from discovery to early clinical trials. A central and timely theme of the network is the development of the unique concept of co-usage of vaccines and microbicides. This review, prepared by the PhD students of the network captures much of the research ongoing between the partners. The network is in its 5th year and involves over 50 institutions from 13 European countries together with 3 industrial partners; GSK, Novartis and Sanofi-Pasteur. EUROPRISE is involved in 31 separate world-wide trials of Vaccines and Microbicides including 6 in African countries (Tanzania, Mozambique, South Africa, Kenya, Malawi, Rwanda), and is directly supporting clinical trials including MABGEL, a gp140-hsp70 conjugate trial and HIVIS, vaccine trials in Europe and Africa.

Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans.

The threat posed by the 2009 pandemic H1N1 virus emphasized the need for new influenza A virus vaccines inducing a broad cross-protective immune response for use in both humans and pigs. An effective and broad influenza vaccine for pigs would greatly benefit the pork industry and contribute to public health by diminishing the risk of emerging highly pathogenic reassortants. Current inactivated protein vaccines against swine influenza produce only short-lived immunity and have no efficacy against heterologous strains. DNA vaccines are a potential alternative with advantages such as the induction of cellular and humoral immunity, inherent safety and rapid production time. We have previously developed a DNA vaccine encoding selected influenza proteins of pandemic origin and demonstrated broad protective immune responses in ferrets and pigs. In this study, we evaluated our DNA vaccine expressed by next-generation vectors. These new vectors can improve gene expression, but they are also efficiently produced on large scales and comply with regulatory guidelines by avoiding antibiotic resistance genes. In addition, a new needle-free delivery of the vaccine, convenient for mass vaccinations, was compared with intradermal needle injection followed by electroporation. We report that when our DNA vaccine is expressed by the new vectors and delivered to the skin with the needle-free device in the rabbit model, it can elicit an antibody response with the same titers as a conventional vector with intradermal electroporation. The needle-free delivery is already in use for traditional protein vaccines in pigs but should be considered as a practical alternative for the mass administration of broadly protective influenza DNA vaccines.

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART).

Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.

A polyvalent influenza DNA vaccine applied by needle-free intradermal delivery induces cross-reactive humoral and cellular immune responses in pigs

Background Pigs are natural hosts for influenza A viruses, and the infection is widely prevalent in swine herds throughout the world. Current commercial influenza vaccines for pigs induce a narrow immune response and are not very effective against antigenically diverse viruses. To control influenza in pigs, the development of more effective swine influenza vaccines inducing broader cross-protective immune responses is needed. Previously, we have shown that a polyvalent influenza DNA vaccine using vectors containing antibiotic resistance genes induced a broadly protective immune response in pigs and ferrets using intradermal injection followed by electroporation. However, this vaccination approach is not practical in large swine herds, and DNA vaccine vectors containing antibiotic resistance genes are undesirable. Objectives To investigate the immunogenicity of an optimized version of our preceding polyvalent DNA vaccine, characterized by a next-generation expression vector without antibiotic resistance markers and delivered by a convenient needle-free intradermal application approach. Methods The humoral and cellular immune responses induced by three different doses of the optimized DNA vaccine were evaluated in groups of five to six pigs. The DNA vaccine consisted of six selected influenza genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase. Results Needle-free vaccination of growing pigs with the optimized DNA vaccine resulted in specific, dose-dependent immunity down to the lowest dose (200 μg DNA/vaccination). Both the antibody-mediated and the recall lymphocyte immune responses demonstrated high reactivity against vaccine-specific strains and cross-reactivity to vaccine-heterologous strains. Conclusion The results suggest that polyvalent DNA influenza vaccination may provide a strong tool for broad protection against swine influenza strains threatening animal as well as public health. In addition, the needle-free administration technique used for this DNA vaccine will provide an easy and practical approach for the large-scale vaccination of swine.

The evolution of HIV-1 interactions with coreceptors and mannose C-type lectin receptors.

The phenotype of human immunodeficiency virus type 1 (HIV-1) commonly evolves between and within infected individuals, at virus transmission, and during disease progression. This evolution includes altered interactions between the virus and its coreceptors, i.e., chemokine receptors, as well as mannose C-type lectin receptors (CLRs). Transmitted/founder viruses are predominantly restricted to CCR5, whereas the subsequent intrapatient evolution of HIV-1 coreceptor use during progressive disease can be subdivided into two distinct pathways. Accordingly, the CCR5-restricted virus population is either gradually replaced by virus variants able to use CXCR4 or evolves toward an altered, more flexible use of CCR5. Despite a strong dependency on these coreceptors for host cell entry, HIV-1 also interacts with other cell surface molecules during target cell attachment, including the CLRs. The virus interaction with the CLRs may result either in the efficient transfer of virus to CD4(+) T cells or in the degradation of the virus in endosomal compartments. The determinants of the diverse outcomes depend on which CLR is engaged and also on the glycan makeup of the envelope glycoproteins, which may evolve with the strength of the immune pressure during the disease course. With the current clinical introduction of CCR5 antagonists and the development of additional entry inhibitors, knowledge on the evolution and baseline characteristics of HIV-1 interactions with coreceptor and CLR interactions may play important roles for individualized and optimized treatment strategies. This review summarizes our current understanding of the evolution of HIV-1 interactions with these receptors.

R5 HIV-1 with efficient DC-SIGN use is not selected for early after birth in vertically infected children.

The binding of human immunodeficiency virus (HIV) to C-type lectin receptors may result in either enhanced trans-infection of T-cells or virus degradation. We have investigated the efficacy of HIV-1 utilization of DC-SIGN, a C-type lectin receptor, in the setting of intrauterine or intrapartum mother-to-child transmission (MTCT). Viruses isolated from HIV-1-infected mothers at delivery and from their vertically infected children both shortly after birth and later during the progression of the disease were analysed for their use of DC-SIGN, binding and ability to trans-infect. DC-SIGN use of a childs earlier virus isolate tended to be reduced as compared with that of the corresponding maternal isolate. Furthermore, the childrens later isolate displayed enhanced DC-SIGN utilization compared with that of the corresponding earlier virus. These results were also supported in head-to-head competition assays and suggest that HIV-1 variants displaying efficient DC-SIGN use are not selected for during intrauterine or intrapartum MTCT. However, viruses with increased DC-SIGN use may evolve later in paediatric HIV-1 infections.

Protective effect of a polyvalent influenza DNA vaccine in pigs

Background Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity. Objectives To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study. Methods By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500 μg and 800 μg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus. Results When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800 μg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500 μg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) − as well as cross-reactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dose-dependent manner. Conclusion The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination.

Increased humoral immunity by DNA vaccination using an α-tocopherol-based adjuvant

ABSTRACT DNA vaccines induce broad immunity, which involves both humoral and strong cellular immunity, and can be rapidly designed for novel or evolving pathogens such as influenza. However, the humoral immunogenicity in humans and higher animals has been suboptimal compared with that of traditional vaccine approaches. We tested whether the emulsion-based and α-tocopherol containing adjuvant Diluvac Forte® has the ability to enhance the immunogenicity of a naked DNA vaccine (i.e., plasmid DNA). As a model vaccine, we used plasmids encoding both a surface-exposed viral glycoprotein (hemagglutinin) and an internal non-glycosylated nucleoprotein in the Th1/Th2 balanced CB6F1 mouse model. The naked DNA (50 µg) was premixed at a 1:1 volume/volume ratio with Diluvac Forte®, an emulsion containing different concentrations of α-tocopherol, the emulsion alone or endotoxin-free phosphate-buffered saline (PBS). The animals received 2 intracutaneous immunizations spaced 3 weeks apart. When combined with Diluvac Forte® or the emulsion containing α-tocopherol, the DNA vaccine induced a more potent and balanced immunoglobulin G (IgG)1 and IgG2c response, and both IgG subclass responses were significantly enhanced by the adjuvant. The DNA vaccine also induced CD4+ and CD8+ vaccine-specific T cells; however, the adjuvant did not exert a significant impact. We concluded that the emulsion-based adjuvant Diluvac Forte® enhanced the immunogenicity of a naked DNA vaccine encoding influenza proteins and that the adjuvant constituent α-tocopherol plays an important role in this immunogenicity. This induction of a potent and balanced humoral response without impairment of cellular immunity constitutes an important advancement toward effective DNA vaccines.