Microbiol Immunol 2014; 58: 219–226 doi: 10.1111/1348-0421.12140
ORIGINAL ARTICLE
Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus Laura Lazo1, Alienys Izquierdo2, Edith Suzarte1, Lázaro Gil1, Iris Valdés1, Ernesto Marcos1, Mayling Álvarez2, Yaremis Romero1, María Guadalupe Guzmán2, Gerardo Guillén1 and Lisset Hermida Cruz1 1
Vaccines Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Playa, Havana 11 600, Cuba and 2Virology Department, Tropical Medicine Institute “Pedro Kourí,” Pan American Health Organization/World Health Organization Collaborating Center for the Study of Dengue and its Vector, P.O. Box Marianao 13, Havana 11 600, Cuba
ABSTRACT A dengue vaccine must induce protective immunity against the four serotypes of the virus. Our group has developed chimeric proteins consisting of the protein P64k from Neisseria meningitidis and the domain III from the four viral envelope proteins. In this study, the immunogenicity of a tetravalent vaccine formulation using aluminum hydroxide as adjuvant was evaluated in mice. After three doses, neutralizing antibody titers were detected against the four viral serotypes, the lowest seroconversion rate being against dengue virus serotype 4. One month after the last dose, immunized animals were challenged with infective virus, and partial but statistically significant protection was found to have been achieved. Based on these results, further studies in mice and non-human primates using this tetravalent formulation in a prime-boost strategy with attenuated viruses are strongly recommended. Key words
dengue, domain III, vaccine, virus.
Dengue fever is caused by any of the four serotypes of dengue virus (DV1–DV4). Infection with one serotype confers life-long immunity against the infecting virus, whereas secondary heterotypic infection can cause severe forms of the disease (1). Therefore an effective vaccine against this pathogen requires a tetravalent formulation. In dengue virus, the envelope protein is the major constituent of the viral envelope and the main target for neutralizing antibodies. Its C-terminal domain (domain III) has been extensively evaluated as a component of vaccine candidates (2–5) because it is likely involved in receptor recognition (6); it is targeted by most mouse monoclonal antibodies strongly neutralizing DVs (7). Our group has developed vaccine candidates based on fusion of domain III from the envelope protein of
dengue virus to the C-terminal region of protein P64k of Neisseria meningitidis (8–10). In the case of the protein corresponding to DV4, a second viral fragment was inserted after the first 45 amino acids of the carrier protein (11). Protein P64k has been used as carrier because it is strongly expressed and strongly immunogenic (12, 13), as well as being safe and having carrier functionality in humans (14). The fusion proteins P64k-domain III used in this study have previously been expressed as inclusion bodies and purified by immobilized metal ion affinity chromatography. The monovalent formulations have been emulsified on Freund adjuvant and evaluated in a mouse model of dengue virus encephalitis (8–11), where they induced neutralizing antibodies and protection. Moreover, the
Correspondence Lisset Hermida Cruz, Vaccines Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Playa, Havana 10 600, Cuba. Tel: þ53 7271 6022 (ext. 7220); fax: þ53 7271 4764; email: [email protected] Received 28 November 2013; revised 16 January 2014; accepted 25 January 2014. List of Abbreviations: DV, dengue virus; GMT, geometric mean titer; LD50, median lethal dose; PBS-T, PBS with 0.05% Tween; tetra PD, tetravalent formulation; USP, United States Pharmacopeia.
© 2014 The Societies and Wiley Publishing Asia Pty Ltd
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L. Lazo et al.
proteins corresponding to serotypes 1 and 2 and emulsified in Freund adjuvant reportedly confer protection in non-human primates (15, 16). In this study, we evaluated a tetravalent formulation (tetra PD) using aluminum hydroxide as adjuvant in mice After three doses, mice developed neutralizing antibodies, cell mediated immunity and partial protection against viral challenge.
MATERIALS AND METHODS Viruses For immunization of positive control groups, preparations of 105 pfu/mL of infective DV1 (Jamaica 288690 strain) (17), DV2 (SB8553) (kindly provided by Dr. Jane Cardosa, University of Malaysia, Sarawak, Malaysia), DV3 (Nicaragua 163/94) (18), and DV4 (Dominica 1981) (19) were used. The immunogen was obtained from cell culture supernatant fluid harvested from infected Vero cells. Preparations from suckling-mice brain infected with DV1 (Hawai strain), DV2 (A15 strain), DV3 (H-87 strain), and DV4 (H241 strain), according to the method of Clarke and Casals (20), were used for viral challenges and ELISA. A similar preparation obtained from the brains of non-inoculated mice was used as negative control. The viral stock for the neutralization assay (D1 Angola [kindly provided by Dr. Robert Shope, University of Texas Medical Branch, Galveston, TX, USA], D2 A15/1981, D3 116/00, and D4 23/00) was obtained from clarified cell culture supernatant fluid harvested from infected Vero cells. Concentrated preparations of each virus (DV1 [Jamaica strain], DV2 [SB8553 strain], DV3 [Nicaragua 163/94], and DV4 [Dominica strain]) were used for in vitro stimulation of mouse splenocytes. These preparations were obtained by centrifuging 100 mL of supernatant from infected Vero cells at 80,000 g for 4 hr at 4 °C, followed by resuspension in 1 mL of PBS. A mock preparation was prepared similarly from the supernatant of uninfected Vero cells. Immunizations and protection assay Groups of 10 7-week-old female Balb/c mice were purchased from the National Center for the Production of Laboratory Animals (Havana, Cuba) and housed in appropriate animal-care facilities during the experimental period. The mice were immunized by the intraperitoneal route on Days 0, 15 and 45. All formulations, except for the positive controls, had a volume of 100 mL and were prepared using Al(OH)3 as adjuvant to a final concentration of 1.44 mg/mL. Negative control mice received 1.44 mg/mL of Al(OH)3. Two doses (250 mL) of 220
infective virus (DV1–DV4) without adjuvant were administered on Days 15 and 45 as positive controls. The mice were bled 15 and 30 days after the last dose and sera collected for further immunological analysis. The groups assayed were as follows: Group 1, 20 mg of PD10 (9); Group 2, 20 mg of PD5 (21); Group 3, 20 mg of PD18 (10); Group 4, 20 mg of PD24 (11); Group 5, tetra PD (80 mg of recombinant protein); Group 6 (positive control), 0.250 mL of DV1 (105 pfu/mL); Group 7 (positive control), 0.250 mL of DV2 (105 pfu/mL); Group 8 (positive control), 0.250 mL of DV3 (105 pfu/mL); Group 9 (positive control), 0.250 mL of DV4 (105 pfu/mL); and Group 10 (placebo), 1.44 mg/mL Al(OH)3. In order to evaluate the protective capacity of the tetra PD against the four serotypes of DV, a second immunization schedule was administered to two groups: a placebo group that received 1.44 mg/mL of Al(OH)3 and a group of animals that was immunized with three doses of the tetra PD formulation by the intraperitoneal route on Days 0, 15 and 45. One month after the third dose, mice from the two groups were divided into four subgroups of 10 mice each. These subgroups were inoculated with 50–100 LD50 of DV1, DV2 or DV4 by an intracranial route. Unfortunately, because lethal neuroadapted strain of DV3 is available, for this serotype the morbidity rather than the mortality was evaluated after brain inoculation. The criteria for evaluating the morbidity of DV3 challenged mice was the presence of symptoms such as hunched posture, ruffled fur, lethargy and paralysis. Mice were observed daily for 21 days. Enzyme-linked immunosorbent assay Amounts of anti-DV antibody were evaluated by an amplified sandwich ELISA system (22). Polystyrene plates with 96 wells (Costar, Cambridge, MA, USA) were coated for 2 hr at 37 °C with 100 mL per well of a mixture of anti-dengue human (IgG) (10 mg/mL) in coating buffer (0.16% Na2CO3, 0.29% NaHCO3, pH 9.5). The plates were then blocked in coating buffer containing 1% BSA for 1 hr at 37 °C and washed three times in PBS-T. Viral antigens from suckling mouse brain infected with DV (100 mL per well) and non-infected suckling mouse brain (as negative control) were incubated overnight at 4 °C. After three washes with PBS-T, 100 mL per well of sera from each group were tested by serial dilutions in PBS-T, starting at 1:100. Plates were incubated for 1 hr at 37 °C and washed as aforementioned. Later, 100 mL per well of anti-mouse IgG–Peroxidase Conjugate (GE Healthcare, Little Chalfont, UK) diluted 1:5,000 were added and the plates incubated for 1 hr at 37 °C. After washing, 100 mL per well of 0.04% substrate (O-phenylenediamine in 2% Na2HPO4 1% citric acid buffer, pH 5.0) were added. The plates were kept for © 2014 The Societies and Wiley Publishing Asia Pty Ltd
Evaluation of a dengue vaccine
30 min at 25 °C, after which the reaction was stopped with 50 mL per well of 12.5% H2SO4. An automated ELISA reader recorded the absorbance at 492 nm. A value was considered positive if it had an absorbance twofold higher than that of the control preparation. Plaque reduction neutralization test Virus replication was determined by plaque formation in a BHK-21 cell culture according to the method of Morens et al. (23). Briefly, Costar 24-well plates were inoculated with culture supernatant and incubated for 4 hr at 37 °C in a 5% CO2 atmosphere. Later, 0.5 mL of 3% mediumviscosity carboxymethylcellulose was added. Plates were incubated at 37 °C in a 5% CO2 atmosphere for 5 days for DV2 and DV4 and 7 days for DV1 and DV3. To visualize the viral plaques the monolayer was dyed with naphtol blue black solution (0.1% naphtol blue black, 0.2 M sodium acetate, 6% acetic acid). The neutralization titer was defined as the dilution yielding a 50% reduction in the maximum number of pfus. Cell culture and cytokine detection One month after the last dose, four mice per group were splenectomized under aseptic conditions. The splenocytes were processed as described elsewhere (22). The cells (2 105 cells per well) were cultured in 96-well round bottom plates with the antigens (DV and mock preparation). Concanavalin A (ConA; Sigma, St. Louis, MO, USA) was used as positive control. The culture supernatants of splenocytes previously stimulated with DV were analyzed in duplicate for INF-g concentrations by ELISA using monoclonal antibody pairs (INFg; Mabtech, Nacía, Sweden). The ELISA protocol recommended by the manufacturers was used. The cut-off for each serotype was defined as twice the value of the placebo mean.
were expressed as inclusion bodies in Escherichia coli and subjected to denaturation–renaturation. The semi-purification process yielded proteins of >80% purity. The endotoxincontent was 100 52 96 66 >100 58 32 72 100%
>100 >100 >100 >100 28 >100 >100 91 100%
26 18 20 53 >100 30 17 38 100%
ORIGINAL ARTICLE
Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus Laura Lazo1, Alienys Izquierdo2, Edith Suzarte1, Lázaro Gil1, Iris Valdés1, Ernesto Marcos1, Mayling Álvarez2, Yaremis Romero1, María Guadalupe Guzmán2, Gerardo Guillén1 and Lisset Hermida Cruz1 1
Vaccines Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Playa, Havana 11 600, Cuba and 2Virology Department, Tropical Medicine Institute “Pedro Kourí,” Pan American Health Organization/World Health Organization Collaborating Center for the Study of Dengue and its Vector, P.O. Box Marianao 13, Havana 11 600, Cuba
ABSTRACT A dengue vaccine must induce protective immunity against the four serotypes of the virus. Our group has developed chimeric proteins consisting of the protein P64k from Neisseria meningitidis and the domain III from the four viral envelope proteins. In this study, the immunogenicity of a tetravalent vaccine formulation using aluminum hydroxide as adjuvant was evaluated in mice. After three doses, neutralizing antibody titers were detected against the four viral serotypes, the lowest seroconversion rate being against dengue virus serotype 4. One month after the last dose, immunized animals were challenged with infective virus, and partial but statistically significant protection was found to have been achieved. Based on these results, further studies in mice and non-human primates using this tetravalent formulation in a prime-boost strategy with attenuated viruses are strongly recommended. Key words
dengue, domain III, vaccine, virus.
Dengue fever is caused by any of the four serotypes of dengue virus (DV1–DV4). Infection with one serotype confers life-long immunity against the infecting virus, whereas secondary heterotypic infection can cause severe forms of the disease (1). Therefore an effective vaccine against this pathogen requires a tetravalent formulation. In dengue virus, the envelope protein is the major constituent of the viral envelope and the main target for neutralizing antibodies. Its C-terminal domain (domain III) has been extensively evaluated as a component of vaccine candidates (2–5) because it is likely involved in receptor recognition (6); it is targeted by most mouse monoclonal antibodies strongly neutralizing DVs (7). Our group has developed vaccine candidates based on fusion of domain III from the envelope protein of
dengue virus to the C-terminal region of protein P64k of Neisseria meningitidis (8–10). In the case of the protein corresponding to DV4, a second viral fragment was inserted after the first 45 amino acids of the carrier protein (11). Protein P64k has been used as carrier because it is strongly expressed and strongly immunogenic (12, 13), as well as being safe and having carrier functionality in humans (14). The fusion proteins P64k-domain III used in this study have previously been expressed as inclusion bodies and purified by immobilized metal ion affinity chromatography. The monovalent formulations have been emulsified on Freund adjuvant and evaluated in a mouse model of dengue virus encephalitis (8–11), where they induced neutralizing antibodies and protection. Moreover, the
Correspondence Lisset Hermida Cruz, Vaccines Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Playa, Havana 10 600, Cuba. Tel: þ53 7271 6022 (ext. 7220); fax: þ53 7271 4764; email: [email protected] Received 28 November 2013; revised 16 January 2014; accepted 25 January 2014. List of Abbreviations: DV, dengue virus; GMT, geometric mean titer; LD50, median lethal dose; PBS-T, PBS with 0.05% Tween; tetra PD, tetravalent formulation; USP, United States Pharmacopeia.
© 2014 The Societies and Wiley Publishing Asia Pty Ltd
219
L. Lazo et al.
proteins corresponding to serotypes 1 and 2 and emulsified in Freund adjuvant reportedly confer protection in non-human primates (15, 16). In this study, we evaluated a tetravalent formulation (tetra PD) using aluminum hydroxide as adjuvant in mice After three doses, mice developed neutralizing antibodies, cell mediated immunity and partial protection against viral challenge.
MATERIALS AND METHODS Viruses For immunization of positive control groups, preparations of 105 pfu/mL of infective DV1 (Jamaica 288690 strain) (17), DV2 (SB8553) (kindly provided by Dr. Jane Cardosa, University of Malaysia, Sarawak, Malaysia), DV3 (Nicaragua 163/94) (18), and DV4 (Dominica 1981) (19) were used. The immunogen was obtained from cell culture supernatant fluid harvested from infected Vero cells. Preparations from suckling-mice brain infected with DV1 (Hawai strain), DV2 (A15 strain), DV3 (H-87 strain), and DV4 (H241 strain), according to the method of Clarke and Casals (20), were used for viral challenges and ELISA. A similar preparation obtained from the brains of non-inoculated mice was used as negative control. The viral stock for the neutralization assay (D1 Angola [kindly provided by Dr. Robert Shope, University of Texas Medical Branch, Galveston, TX, USA], D2 A15/1981, D3 116/00, and D4 23/00) was obtained from clarified cell culture supernatant fluid harvested from infected Vero cells. Concentrated preparations of each virus (DV1 [Jamaica strain], DV2 [SB8553 strain], DV3 [Nicaragua 163/94], and DV4 [Dominica strain]) were used for in vitro stimulation of mouse splenocytes. These preparations were obtained by centrifuging 100 mL of supernatant from infected Vero cells at 80,000 g for 4 hr at 4 °C, followed by resuspension in 1 mL of PBS. A mock preparation was prepared similarly from the supernatant of uninfected Vero cells. Immunizations and protection assay Groups of 10 7-week-old female Balb/c mice were purchased from the National Center for the Production of Laboratory Animals (Havana, Cuba) and housed in appropriate animal-care facilities during the experimental period. The mice were immunized by the intraperitoneal route on Days 0, 15 and 45. All formulations, except for the positive controls, had a volume of 100 mL and were prepared using Al(OH)3 as adjuvant to a final concentration of 1.44 mg/mL. Negative control mice received 1.44 mg/mL of Al(OH)3. Two doses (250 mL) of 220
infective virus (DV1–DV4) without adjuvant were administered on Days 15 and 45 as positive controls. The mice were bled 15 and 30 days after the last dose and sera collected for further immunological analysis. The groups assayed were as follows: Group 1, 20 mg of PD10 (9); Group 2, 20 mg of PD5 (21); Group 3, 20 mg of PD18 (10); Group 4, 20 mg of PD24 (11); Group 5, tetra PD (80 mg of recombinant protein); Group 6 (positive control), 0.250 mL of DV1 (105 pfu/mL); Group 7 (positive control), 0.250 mL of DV2 (105 pfu/mL); Group 8 (positive control), 0.250 mL of DV3 (105 pfu/mL); Group 9 (positive control), 0.250 mL of DV4 (105 pfu/mL); and Group 10 (placebo), 1.44 mg/mL Al(OH)3. In order to evaluate the protective capacity of the tetra PD against the four serotypes of DV, a second immunization schedule was administered to two groups: a placebo group that received 1.44 mg/mL of Al(OH)3 and a group of animals that was immunized with three doses of the tetra PD formulation by the intraperitoneal route on Days 0, 15 and 45. One month after the third dose, mice from the two groups were divided into four subgroups of 10 mice each. These subgroups were inoculated with 50–100 LD50 of DV1, DV2 or DV4 by an intracranial route. Unfortunately, because lethal neuroadapted strain of DV3 is available, for this serotype the morbidity rather than the mortality was evaluated after brain inoculation. The criteria for evaluating the morbidity of DV3 challenged mice was the presence of symptoms such as hunched posture, ruffled fur, lethargy and paralysis. Mice were observed daily for 21 days. Enzyme-linked immunosorbent assay Amounts of anti-DV antibody were evaluated by an amplified sandwich ELISA system (22). Polystyrene plates with 96 wells (Costar, Cambridge, MA, USA) were coated for 2 hr at 37 °C with 100 mL per well of a mixture of anti-dengue human (IgG) (10 mg/mL) in coating buffer (0.16% Na2CO3, 0.29% NaHCO3, pH 9.5). The plates were then blocked in coating buffer containing 1% BSA for 1 hr at 37 °C and washed three times in PBS-T. Viral antigens from suckling mouse brain infected with DV (100 mL per well) and non-infected suckling mouse brain (as negative control) were incubated overnight at 4 °C. After three washes with PBS-T, 100 mL per well of sera from each group were tested by serial dilutions in PBS-T, starting at 1:100. Plates were incubated for 1 hr at 37 °C and washed as aforementioned. Later, 100 mL per well of anti-mouse IgG–Peroxidase Conjugate (GE Healthcare, Little Chalfont, UK) diluted 1:5,000 were added and the plates incubated for 1 hr at 37 °C. After washing, 100 mL per well of 0.04% substrate (O-phenylenediamine in 2% Na2HPO4 1% citric acid buffer, pH 5.0) were added. The plates were kept for © 2014 The Societies and Wiley Publishing Asia Pty Ltd
Evaluation of a dengue vaccine
30 min at 25 °C, after which the reaction was stopped with 50 mL per well of 12.5% H2SO4. An automated ELISA reader recorded the absorbance at 492 nm. A value was considered positive if it had an absorbance twofold higher than that of the control preparation. Plaque reduction neutralization test Virus replication was determined by plaque formation in a BHK-21 cell culture according to the method of Morens et al. (23). Briefly, Costar 24-well plates were inoculated with culture supernatant and incubated for 4 hr at 37 °C in a 5% CO2 atmosphere. Later, 0.5 mL of 3% mediumviscosity carboxymethylcellulose was added. Plates were incubated at 37 °C in a 5% CO2 atmosphere for 5 days for DV2 and DV4 and 7 days for DV1 and DV3. To visualize the viral plaques the monolayer was dyed with naphtol blue black solution (0.1% naphtol blue black, 0.2 M sodium acetate, 6% acetic acid). The neutralization titer was defined as the dilution yielding a 50% reduction in the maximum number of pfus. Cell culture and cytokine detection One month after the last dose, four mice per group were splenectomized under aseptic conditions. The splenocytes were processed as described elsewhere (22). The cells (2 105 cells per well) were cultured in 96-well round bottom plates with the antigens (DV and mock preparation). Concanavalin A (ConA; Sigma, St. Louis, MO, USA) was used as positive control. The culture supernatants of splenocytes previously stimulated with DV were analyzed in duplicate for INF-g concentrations by ELISA using monoclonal antibody pairs (INFg; Mabtech, Nacía, Sweden). The ELISA protocol recommended by the manufacturers was used. The cut-off for each serotype was defined as twice the value of the placebo mean.
were expressed as inclusion bodies in Escherichia coli and subjected to denaturation–renaturation. The semi-purification process yielded proteins of >80% purity. The endotoxincontent was 100 52 96 66 >100 58 32 72 100%
>100 >100 >100 >100 28 >100 >100 91 100%
26 18 20 53 >100 30 17 38 100%
