- Email: [email protected]
Gene-therapy for malaria prevention
TREPAR-1321; No. of Pages 3
Spotlight
Gene-therapy for malaria prevention Mauricio M. Rodrigues1,2 and Irene S. Soares3 1
Centro de Terapia Celular e Molecular (CTCMol), Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo-Escola Paulista de Medicina, Sa˜o Paulo, SP, Brazil 3 Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil 2
The limited number of tools for malaria prevention and the inability to eradicate the disease have required large investments in vaccine development, as vaccines have been the only foreseeable type of immunoprophylaxis against malaria. An alternative strategy named vectored immunoprophylaxis (VIP) now would allow genetically transduced host cells to assemble and secrete antibodies that neutralize the infectivity of the malaria parasite and prevent disease. Malaria remains a devastating parasitic disease worldwide. The number of people at risk is estimated at more than 3.5 billion individuals, who live primarily in tropical areas. The number of estimated cases is between 135 to 287 million, resulting in a death toll of 473 000 – 789 000 persons per year (http://www.who.int/malaria/ media/world_malaria_report_2013/en/). In most cases, these deaths involve African children under the age of 5 years. Although the disease is most prevalent in Africa due to Plasmodium falciparum infection, malaria transmission also occurs in large areas of South Asia, Latin America, and Oceania. In these areas, in addition to P. falciparum, there is also significant transmission of Plasmodium vivax, representing as much as 65% of the cases in the Americas. Malaria is considered a preventable disease. It is estimated that between 2000 and 2012, malaria incidence was reduced by 25% globally, preventing 42% of the potential deaths (http://www.who.int/malaria/media/world_malaria_ report_2013/en/). One of the main obstacles faced by attempts to improve malaria prevention is that, traditionally, only a few strategies have been used, that is, education, chemotherapy, and vector control using insecticides or elimination of mosquito breeding sites. In the past 20 years, the only addition to these traditional methods has been the use of insecticide-impregnated mosquito bed nets. Because of the limited number of tools for malaria prevention and the inability of these tools to eradicate the disease in many endemic areas, immunoprophylaxis against malaria has been pursued during the past 50 years. These substantial investments led to the first formulation of a vaccine against the deadly P. falciparum parasite. This vaccine is being tested in large-scale Phase III studies in Corresponding author: Rodrigues, M.M. ([email protected]). Keywords: gene therapy; malaria prevention; immune-prophylaxis. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.09.005
African children. The RTS,S vaccine is a fusion protein between portions of the major antigen present on the surface of the infective stages of malaria parasites, the circumsporozoite protein (CSP) and the hepatitis B surface antigen (S), administered in an adjuvant system (AS) containing monophosphoryl lipid A [a detoxified form of lipopolysaccharide (LPS)] and QS21 (purified saponin from Quillaja saponaria) in liposomes [1]. In recent Phase III clinical trials, protective efficacies ranging from 40% to 77% have been reported [2]. Studies on the mechanisms of immunity have yielded the conclusion that the primary mechanistic basis of malaria prevention is the concentration of antibodies directed to the main epitope of the CSP, that is, NANP repetitions [3]. The protective efficacy of anti-CSP immunoglobulin G (IgG) from these RTS,S human vaccinated individuals was evaluated by the administration of three human anti-CSP monoclonal antibodies (mAbs) into humanized uPA-SCID mice before exposure to P. falciparum. Passive transfer of anti-CSP mAbs resulted in a reduction of the liver parasite burden and provided sterilizing immunity against P. falciparum infection when a critical serum concentration was reached [4]. Despite these promising results, a number of problems remain to be solved to improve the efficacy of the vaccine. First, these high antibody concentrations are only achieved by a portion of the vaccinated individuals. In those who achieve these high antibody concentrations, they do not last much longer than two years. By 4 years, the vaccine efficacy is reduced to near baseline [5]. Alternative approaches that can generate and maintain high antibody concentrations for extended periods of time by vaccination remain elusive. Recent developments in an unrelated field may provide a new strategy to promote antibody-mediated immunoprophylaxis without vaccination. This strategy is named vectored immunoprophylaxis. It does not require injecting of the antigen to elicit an immune response. In this strategy, genetically transduced cells of the hosts can assemble and secrete the entire antibody that will fight the invading pathogen. The history behind the development of this new strategy for immunoprophylaxis is linked to past developments in the field of gene therapy. This technique was initially conceived to replace defective genes observed in genetic disorders such as cystic fibrosis and hemophilia. In these cases, the injected foreign DNA would be capable of transducing the individual cells and replacing the defective Trends in Parasitology xx (2014) 1–3
1
TREPAR-1321; No. of Pages 3
Spotlight gene, leading to the synthesis of the protein that the deficient individuals were unable to produce. In subsequent years, the observation that injected foreign DNA could not only lead to the synthesis of proteins but also initiate an immune response has created the new field of genetic vaccination. In these cases, the antigenic protein would be synthesized by the host’s own body and elicit an immune response. For both gene therapy and genetic vaccination, a number of new expression vectors have been created, including plasmidial DNA and replication-incompetent viruses. The rapid evolution of these fields has served as the foundation of the development of yet a third field: vectored immunoprophylaxis (VIP). Adeno-associated virus-based vectors (AAVs) have been selected to develop this new immunoprophylactic approach. These vectors do not integrate into the genome and transduce nonreplicating and long-lived cells in vivo, leading to the expression of the new protein from months to years in their associated tissues. Despite some degree of immune response to the vector-infected cells, most of the transduced cells survive for a long time. The limited packaging capacity of these AAVs has led to a series of modifications of their genome to accommodate larger DNA sequences and, more importantly, to allow the expression of Ig genes. In this context, a dual-promoter AAV is used in which a foot-and-mouth disease virusderived 2A self-processing sequence (F2A) is engineered with a furin cleavage site. Under this configuration, the efficient expression of heavy and light chains is enabled from a single reading frame (reviewed in [6]). This genomic organization has been further improved for high levels of expression by creating a new hybrid promoter, designated CASI, that combines the enhancer of the cytomegalovirus immediate early promoter (CMV), the b-actin promoter, and the enhancer of ubiquitin C (UBC) promoter flanked by a splicing donor and acceptor. Finally, the incorporation of the woodchuck hepatitis virus posttranslational regulatory element (WPRE) and codon optimization of the genes have drastically improved the production of the recombinant antibody [7] (Figure 1). This improved AAV vector was used to express a series of mAbs specific for HIV that have been previously shown to exert viral inhibitory activity in vivo. A single intramuscular injection of recombinant AAV8 could achieve peak antibody production in serum at week 16 and persist for up to 64 weeks. Biological assays in vivo were performed initially by the administration of the recombinant AAV to immunodeficient mice transplanted with human peripheral blood mononuclear cells and challenged intravenously with the NL4-3 strain of HIV. When mice were monitored for CD4 depletion and the level of HIV p24 in tissues, one of the recombinant antibodies provided full protection and another four showed partial protection [7]. Subsequent studies were conducted to test more thoroughly whether VIP could provide immunity to mucosal challenges with different strains of HIV. With a new recombinant AAV expressing the broadly reactive antibody VRC07G54 W, it was possible to provide substantial 2
Trends in Parasitology xxx xxxx, Vol. xxx, No. x
VIP expression vector ITR
CASI
IgG HC
2A
LC
WPRE SV40pA
ITR
Anbody variable regions
Recombinant AAV Intramuscular injecon
Sporozoite neutralizaon
mAb secreon by transduced muscle cells
TRENDS in Parasitology
Figure 1. VIP for malaria prevention. A VIP expression vector can be used to generate recombinant AAV. These viruses can be administered intramuscularly into hosts. Transduced muscle cells will secrete large amounts of recombinant antibodies. They can neutralize the infectivity of malaria sporozoites and prevent the infection of liver cells. Abbreviations: AAV, adeno-associated virus-based vectors; VIP, vectored immunoprophylaxis.
protection to CD4 cells in mucosal tissues. These results suggest that VIP using broadly neutralizing antibodies can protect humanized mice against infection by strains of HIV similar to those responsible for human transmission [8]. Given that effective long-lasting immunity against malaria has not yet been achieved, this unorthodox approach, VIP, was adopted in an attempt to prevent malaria. For this purpose, VIP vectors encoding antibodies specific for the P. falciparum CSP repeats were generated by inserting the variable regions of two mouse mAbs (2A10 and 2C11) into the hIgG framework [9]. mAbs 2A10 and 2C11 were selected based on previous experiments in vivo. When passively transferred to mice, they reduce infection with transgenic Plasmodium berghei sporozoites expressing the central repeat domain of P. falciparum CSP. The vectors 2A10-AAV and 2C11-AAV (1011 genome copies) were injected intramuscularly in C57BL/6 mice. Transduced mice expressed hIgG at 50 to 500 mg/mL in serum, reaching 1 000 mg/mL in a few animals, until 8 weeks, when they were challenged by mosquito bites. Sixty percent of 2A10- and 30% of 2C11-AAV-transduced mice achieved sterile protection against the experimental infection. Compared with the control group, mice that developed parasitemia displayed significant delays in the time to parasitemia. Sterile immunity correlated with
TREPAR-1321; No. of Pages 3
Spotlight 2A10 mAb levels of >1 000 mg/mL. The authors concluded that in a murine model of human P. falciparum infection, a single intramuscular injection of a VIP vector encoding a mAb that neutralizes malaria sporozoites can confer protection from mosquito-transmitted malaria infection [9]. To validate this approach for human use, experiments are being pursued in nonhuman primates to determine effective VIP protocols (e.g., doses and sites of muscle injection). As VIP is a form of AAV-based gene therapy, it is subject to all the safety issues involved in gene therapy. Although these issues are of great concern, it is encouraging that AAV-based gene therapy for genetic diseases has recently been the basis for multiple clinical trials and that intramuscular injection of the AAV1 vector to treat Familial Lipoprotein Lipase deficiency disease has been licensed in Europe [10]. In summary, after more than 50 years with a single strategy of immunoprophylaxis, VIP opens unexpected new avenues for malaria prevention. Acknowledgments This work was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (2012/17060-3 and 2012/13032-5), Instituto Nacional de Cieˆncia e Tecnologia em Vacina (INCTV-CNPq), PNPD (CNPq-CAPES) and CNPq Universal. M.M.R. and I.S.S. are recipients of fellowships from CNPq.
Trends in Parasitology xxx xxxx, Vol. xxx, No. x
Disclaimer statement We would like to disclose the following conflict of interest: M.M.R and I.S.S. are named inventors on patent applications covering Plasmodium vivax vaccine composition.
References 1 Cohen, J. et al. (2010) From the circumsporozoite protein to the RTS, S/ AS candidate vaccine. Hum. Vaccin. 6, 90–96 2 The RTS, S Clinical Trials Partnership (2014) Efficacy and safety of the RTS, S/AS01 malaria vaccine during 18 months after vaccination: a phase 3 randomized, controlled trial in children and young infants at 11 African sites. PLoS Med. 11, e1001685 3 Olotu, A. et al. (2011) Efficacy of RTS, S/AS01E malaria vaccine and exploratory analysis on anti-circumsporozoite antibody titres and protection in children aged 5-17 months in Kenya and Tanzania: a randomised controlled trial. Lancet Infect. Dis. 11, 102–109 4 Foquet, L. et al. (2014) Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection. J. Clin. Invest. 124, 140–144 5 Olotu, A. et al. (2013) Four-year efficacy of RTS, S/AS01E and its interaction with malaria exposure. N. Engl. J. Med. 368, 1111–1120 6 Yang, L. and Wang, P. (2014) Passive immunization against HIV/AIDS by antibody gene transfer. Viruses 6, 428–447 7 Balazs, A.B. et al. (2011) Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature 481, 81–84 8 Balazs, A.B. et al. (2013) Vectored immunoprophylaxis protects humanized mice from mucosal HIV transmission. Nat. Med. 20, 296–300 9 Deal, C. et al. (2014) Vectored antibody gene delivery protects against Plasmodium falciparum sporozoite challenge in mice. Proc. Natl. Acad. Sci. U.S.A. 111, 12528–12532 10 Cressey, D. (2012) Europe nears first approval for gene therapy. Nature http://dx.doi.org/10.1038/nature.2012.11048
3
Spotlight
Gene-therapy for malaria prevention Mauricio M. Rodrigues1,2 and Irene S. Soares3 1
Centro de Terapia Celular e Molecular (CTCMol), Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo-Escola Paulista de Medicina, Sa˜o Paulo, SP, Brazil 3 Departamento de Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de Cieˆncias Farmaceˆuticas, Universidade de Sa˜o Paulo, Sa˜o Paulo, SP, Brazil 2
The limited number of tools for malaria prevention and the inability to eradicate the disease have required large investments in vaccine development, as vaccines have been the only foreseeable type of immunoprophylaxis against malaria. An alternative strategy named vectored immunoprophylaxis (VIP) now would allow genetically transduced host cells to assemble and secrete antibodies that neutralize the infectivity of the malaria parasite and prevent disease. Malaria remains a devastating parasitic disease worldwide. The number of people at risk is estimated at more than 3.5 billion individuals, who live primarily in tropical areas. The number of estimated cases is between 135 to 287 million, resulting in a death toll of 473 000 – 789 000 persons per year (http://www.who.int/malaria/ media/world_malaria_report_2013/en/). In most cases, these deaths involve African children under the age of 5 years. Although the disease is most prevalent in Africa due to Plasmodium falciparum infection, malaria transmission also occurs in large areas of South Asia, Latin America, and Oceania. In these areas, in addition to P. falciparum, there is also significant transmission of Plasmodium vivax, representing as much as 65% of the cases in the Americas. Malaria is considered a preventable disease. It is estimated that between 2000 and 2012, malaria incidence was reduced by 25% globally, preventing 42% of the potential deaths (http://www.who.int/malaria/media/world_malaria_ report_2013/en/). One of the main obstacles faced by attempts to improve malaria prevention is that, traditionally, only a few strategies have been used, that is, education, chemotherapy, and vector control using insecticides or elimination of mosquito breeding sites. In the past 20 years, the only addition to these traditional methods has been the use of insecticide-impregnated mosquito bed nets. Because of the limited number of tools for malaria prevention and the inability of these tools to eradicate the disease in many endemic areas, immunoprophylaxis against malaria has been pursued during the past 50 years. These substantial investments led to the first formulation of a vaccine against the deadly P. falciparum parasite. This vaccine is being tested in large-scale Phase III studies in Corresponding author: Rodrigues, M.M. ([email protected]). Keywords: gene therapy; malaria prevention; immune-prophylaxis. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.09.005
African children. The RTS,S vaccine is a fusion protein between portions of the major antigen present on the surface of the infective stages of malaria parasites, the circumsporozoite protein (CSP) and the hepatitis B surface antigen (S), administered in an adjuvant system (AS) containing monophosphoryl lipid A [a detoxified form of lipopolysaccharide (LPS)] and QS21 (purified saponin from Quillaja saponaria) in liposomes [1]. In recent Phase III clinical trials, protective efficacies ranging from 40% to 77% have been reported [2]. Studies on the mechanisms of immunity have yielded the conclusion that the primary mechanistic basis of malaria prevention is the concentration of antibodies directed to the main epitope of the CSP, that is, NANP repetitions [3]. The protective efficacy of anti-CSP immunoglobulin G (IgG) from these RTS,S human vaccinated individuals was evaluated by the administration of three human anti-CSP monoclonal antibodies (mAbs) into humanized uPA-SCID mice before exposure to P. falciparum. Passive transfer of anti-CSP mAbs resulted in a reduction of the liver parasite burden and provided sterilizing immunity against P. falciparum infection when a critical serum concentration was reached [4]. Despite these promising results, a number of problems remain to be solved to improve the efficacy of the vaccine. First, these high antibody concentrations are only achieved by a portion of the vaccinated individuals. In those who achieve these high antibody concentrations, they do not last much longer than two years. By 4 years, the vaccine efficacy is reduced to near baseline [5]. Alternative approaches that can generate and maintain high antibody concentrations for extended periods of time by vaccination remain elusive. Recent developments in an unrelated field may provide a new strategy to promote antibody-mediated immunoprophylaxis without vaccination. This strategy is named vectored immunoprophylaxis. It does not require injecting of the antigen to elicit an immune response. In this strategy, genetically transduced cells of the hosts can assemble and secrete the entire antibody that will fight the invading pathogen. The history behind the development of this new strategy for immunoprophylaxis is linked to past developments in the field of gene therapy. This technique was initially conceived to replace defective genes observed in genetic disorders such as cystic fibrosis and hemophilia. In these cases, the injected foreign DNA would be capable of transducing the individual cells and replacing the defective Trends in Parasitology xx (2014) 1–3
1
TREPAR-1321; No. of Pages 3
Spotlight gene, leading to the synthesis of the protein that the deficient individuals were unable to produce. In subsequent years, the observation that injected foreign DNA could not only lead to the synthesis of proteins but also initiate an immune response has created the new field of genetic vaccination. In these cases, the antigenic protein would be synthesized by the host’s own body and elicit an immune response. For both gene therapy and genetic vaccination, a number of new expression vectors have been created, including plasmidial DNA and replication-incompetent viruses. The rapid evolution of these fields has served as the foundation of the development of yet a third field: vectored immunoprophylaxis (VIP). Adeno-associated virus-based vectors (AAVs) have been selected to develop this new immunoprophylactic approach. These vectors do not integrate into the genome and transduce nonreplicating and long-lived cells in vivo, leading to the expression of the new protein from months to years in their associated tissues. Despite some degree of immune response to the vector-infected cells, most of the transduced cells survive for a long time. The limited packaging capacity of these AAVs has led to a series of modifications of their genome to accommodate larger DNA sequences and, more importantly, to allow the expression of Ig genes. In this context, a dual-promoter AAV is used in which a foot-and-mouth disease virusderived 2A self-processing sequence (F2A) is engineered with a furin cleavage site. Under this configuration, the efficient expression of heavy and light chains is enabled from a single reading frame (reviewed in [6]). This genomic organization has been further improved for high levels of expression by creating a new hybrid promoter, designated CASI, that combines the enhancer of the cytomegalovirus immediate early promoter (CMV), the b-actin promoter, and the enhancer of ubiquitin C (UBC) promoter flanked by a splicing donor and acceptor. Finally, the incorporation of the woodchuck hepatitis virus posttranslational regulatory element (WPRE) and codon optimization of the genes have drastically improved the production of the recombinant antibody [7] (Figure 1). This improved AAV vector was used to express a series of mAbs specific for HIV that have been previously shown to exert viral inhibitory activity in vivo. A single intramuscular injection of recombinant AAV8 could achieve peak antibody production in serum at week 16 and persist for up to 64 weeks. Biological assays in vivo were performed initially by the administration of the recombinant AAV to immunodeficient mice transplanted with human peripheral blood mononuclear cells and challenged intravenously with the NL4-3 strain of HIV. When mice were monitored for CD4 depletion and the level of HIV p24 in tissues, one of the recombinant antibodies provided full protection and another four showed partial protection [7]. Subsequent studies were conducted to test more thoroughly whether VIP could provide immunity to mucosal challenges with different strains of HIV. With a new recombinant AAV expressing the broadly reactive antibody VRC07G54 W, it was possible to provide substantial 2
Trends in Parasitology xxx xxxx, Vol. xxx, No. x
VIP expression vector ITR
CASI
IgG HC
2A
LC
WPRE SV40pA
ITR
Anbody variable regions
Recombinant AAV Intramuscular injecon
Sporozoite neutralizaon
mAb secreon by transduced muscle cells
TRENDS in Parasitology
Figure 1. VIP for malaria prevention. A VIP expression vector can be used to generate recombinant AAV. These viruses can be administered intramuscularly into hosts. Transduced muscle cells will secrete large amounts of recombinant antibodies. They can neutralize the infectivity of malaria sporozoites and prevent the infection of liver cells. Abbreviations: AAV, adeno-associated virus-based vectors; VIP, vectored immunoprophylaxis.
protection to CD4 cells in mucosal tissues. These results suggest that VIP using broadly neutralizing antibodies can protect humanized mice against infection by strains of HIV similar to those responsible for human transmission [8]. Given that effective long-lasting immunity against malaria has not yet been achieved, this unorthodox approach, VIP, was adopted in an attempt to prevent malaria. For this purpose, VIP vectors encoding antibodies specific for the P. falciparum CSP repeats were generated by inserting the variable regions of two mouse mAbs (2A10 and 2C11) into the hIgG framework [9]. mAbs 2A10 and 2C11 were selected based on previous experiments in vivo. When passively transferred to mice, they reduce infection with transgenic Plasmodium berghei sporozoites expressing the central repeat domain of P. falciparum CSP. The vectors 2A10-AAV and 2C11-AAV (1011 genome copies) were injected intramuscularly in C57BL/6 mice. Transduced mice expressed hIgG at 50 to 500 mg/mL in serum, reaching 1 000 mg/mL in a few animals, until 8 weeks, when they were challenged by mosquito bites. Sixty percent of 2A10- and 30% of 2C11-AAV-transduced mice achieved sterile protection against the experimental infection. Compared with the control group, mice that developed parasitemia displayed significant delays in the time to parasitemia. Sterile immunity correlated with
TREPAR-1321; No. of Pages 3
Spotlight 2A10 mAb levels of >1 000 mg/mL. The authors concluded that in a murine model of human P. falciparum infection, a single intramuscular injection of a VIP vector encoding a mAb that neutralizes malaria sporozoites can confer protection from mosquito-transmitted malaria infection [9]. To validate this approach for human use, experiments are being pursued in nonhuman primates to determine effective VIP protocols (e.g., doses and sites of muscle injection). As VIP is a form of AAV-based gene therapy, it is subject to all the safety issues involved in gene therapy. Although these issues are of great concern, it is encouraging that AAV-based gene therapy for genetic diseases has recently been the basis for multiple clinical trials and that intramuscular injection of the AAV1 vector to treat Familial Lipoprotein Lipase deficiency disease has been licensed in Europe [10]. In summary, after more than 50 years with a single strategy of immunoprophylaxis, VIP opens unexpected new avenues for malaria prevention. Acknowledgments This work was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (2012/17060-3 and 2012/13032-5), Instituto Nacional de Cieˆncia e Tecnologia em Vacina (INCTV-CNPq), PNPD (CNPq-CAPES) and CNPq Universal. M.M.R. and I.S.S. are recipients of fellowships from CNPq.
Trends in Parasitology xxx xxxx, Vol. xxx, No. x
Disclaimer statement We would like to disclose the following conflict of interest: M.M.R and I.S.S. are named inventors on patent applications covering Plasmodium vivax vaccine composition.
References 1 Cohen, J. et al. (2010) From the circumsporozoite protein to the RTS, S/ AS candidate vaccine. Hum. Vaccin. 6, 90–96 2 The RTS, S Clinical Trials Partnership (2014) Efficacy and safety of the RTS, S/AS01 malaria vaccine during 18 months after vaccination: a phase 3 randomized, controlled trial in children and young infants at 11 African sites. PLoS Med. 11, e1001685 3 Olotu, A. et al. (2011) Efficacy of RTS, S/AS01E malaria vaccine and exploratory analysis on anti-circumsporozoite antibody titres and protection in children aged 5-17 months in Kenya and Tanzania: a randomised controlled trial. Lancet Infect. Dis. 11, 102–109 4 Foquet, L. et al. (2014) Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection. J. Clin. Invest. 124, 140–144 5 Olotu, A. et al. (2013) Four-year efficacy of RTS, S/AS01E and its interaction with malaria exposure. N. Engl. J. Med. 368, 1111–1120 6 Yang, L. and Wang, P. (2014) Passive immunization against HIV/AIDS by antibody gene transfer. Viruses 6, 428–447 7 Balazs, A.B. et al. (2011) Antibody-based protection against HIV infection by vectored immunoprophylaxis. Nature 481, 81–84 8 Balazs, A.B. et al. (2013) Vectored immunoprophylaxis protects humanized mice from mucosal HIV transmission. Nat. Med. 20, 296–300 9 Deal, C. et al. (2014) Vectored antibody gene delivery protects against Plasmodium falciparum sporozoite challenge in mice. Proc. Natl. Acad. Sci. U.S.A. 111, 12528–12532 10 Cressey, D. (2012) Europe nears first approval for gene therapy. Nature http://dx.doi.org/10.1038/nature.2012.11048
3