Expression-system-dependent modulation of HIV-1 envelope glycoprotein antigenicity and immunogenicity.
Kong Leopold,Sheppard Neil C,Stewart-Jones Guillaume B E,Robson Cynthia L,Chen Hongying,Xu Xiaodong,Krashias George,Bonomelli Camille,Scanlan Christopher N,Kwong Peter D,Jeffs Simon A,Jones Ian M,Sattentau Quentin J
Journal of molecular biology
Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure-function studies and for vaccine use.
10.1016/j.jmb.2010.08.033
Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice.
Li Dapeng,von Schaewen Markus,Wang Xuesong,Tao Wanyin,Zhang Yunfang,Li Li,Heller Brigitte,Hrebikova Gabriela,Deng Qiang,Ploss Alexander,Zhong Jin,Huang Zhong
Journal of virology
Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among the genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possesses different glycosylation patterns and is more immunogenic, as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate, but also highlight the importance of glycosylation patterns in developing subunit viral vaccines. IMPORTANCE:A prophylactic vaccine with high efficacy and low cost is urgently needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Here, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus, our work identified a promising broadly protective HCV vaccine candidate that should be considered for further preclinical and clinical development.
10.1128/JVI.01462-16
Limited Evidence for a Relationship between HIV-1 Glycan Shield Features in Early Infection and the Development of Neutralization Breadth.
Li Yifan,Bai Hongjun,Sanders-Buell Eric,Dussupt Vincent,Townsley Samantha,Donofrio Gina,Bose Meera,O'Sullivan Anne Marie,Kibuuka Hannah,Maganga Lucas,Nitayaphan Sorachai,Kosgei Josphat,Pitisuttithum Punnee,Rerks-Ngarm Supachai,Eller Leigh Anne,Michael Nelson L,Robb Merlin L,Ake Julie,Vasan Sandhya,Tovanabutra Sodsai,Krebs Shelly J,Rolland Morgane
Journal of virology
Identifying whether viral features present in acute HIV-1 infection predetermine the development of neutralization breadth is critical to vaccine design. Incorporating such features in vaccine antigens could initiate cross-reactive antibody responses that could sufficiently protect vaccinees from HIV-1 infection despite the uniqueness of each founder virus. To understand the relationship between Env determinants and the development of neutralization breadth, we focused on 197 individuals enrolled in two cohorts in Thailand and East Africa (RV144 and RV217) and followed since their diagnosis in acute or early HIV-1 infection. We analyzed the distribution of variable loop lengths and glycans, as well as the predicted density of the glycan shield, and compared these envelope features to the neutralization breadth data obtained 3 years after infection ( = 121). Our study revealed limited evidence for glycan shield features that associate with the development of neutralization breadth. While the glycan shield tended to be denser in participants who subsequently developed breadth, no significant relationship was found between the size of glycan holes and the development of neutralization breadth. The parallel analysis of 3,000 independent Env sequences showed no evidence of directional evolution of glycan shield features since the beginning of the epidemic. Together, our results highlight that glycan shield features in acute and early HIV-1 infection may not play a role determinant enough to dictate the development of neutralization breadth and instead suggest that the glycan shield's reactive properties that are associated with immune evasion may have a greater impact. A major goal of HIV-1 vaccine research is to design vaccine candidates that elicit potent broadly neutralizing antibodies (bNAbs). Different viral features have been associated with the development of bNAbs, including the glycan shield on the surface of the HIV-1 Envelope (Env). Here, we analyzed data from two cohorts of individuals who were followed from early infection to several years after infection spanning multiple HIV-1 subtypes. We compared Env glycan features in HIV-1 sequences obtained in early infection to the potency and breadth of neutralizing antibodies measured 1 to 3 years after infection. We found limited evidence of glycan shield properties that associate with the development of neutralization breadth in these cohorts. These results may have important implications for antigen design in future vaccine strategies and emphasize that HIV-1 vaccines will need to rely on a complex set of properties to elicit neutralization breadth.
10.1128/JVI.00797-21
Quantification of the Impact of the HIV-1-Glycan Shield on Antibody Elicitation.
Zhou Tongqing,Doria-Rose Nicole A,Cheng Cheng,Stewart-Jones Guillaume B E,Chuang Gwo-Yu,Chambers Michael,Druz Aliaksandr,Geng Hui,McKee Krisha,Kwon Young Do,O'Dell Sijy,Sastry Mallika,Schmidt Stephen D,Xu Kai,Chen Lei,Chen Rita E,Louder Mark K,Pancera Marie,Wanninger Timothy G,Zhang Baoshan,Zheng Anqi,Farney S Katie,Foulds Kathryn E,Georgiev Ivelin S,Joyce M Gordon,Lemmin Thomas,Narpala Sandeep,Rawi Reda,Soto Cinque,Todd John-Paul,Shen Chen-Hsiang,Tsybovsky Yaroslav,Yang Yongping,Zhao Peng,Haynes Barton F,Stamatatos Leonidas,Tiemeyer Michael,Wells Lance,Scorpio Diana G,Shapiro Lawrence,McDermott Adrian B,Mascola John R,Kwong Peter D
Cell reports
While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here, we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses but potent CD4-supersite neutralization (titers 1: >1,000,000 against four-glycan-deleted autologous viruses with over 90% breadth against four-glycan-deleted heterologous strains exhibiting tier 2 neutralization character). To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID) proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.
10.1016/j.celrep.2017.04.013
Egg-based influenza split virus vaccine with monoglycosylation induces cross-strain protection against influenza virus infections.
Tseng Yung-Chieh,Wu Chia-Yu,Liu Ming-Liang,Chen Ting-Hua,Chiang Wan-Ling,Yu Yueh-Hsiang,Jan Jia-Tsrong,Lin Kuo-I,Wong Chi-Huey,Ma Che
Proceedings of the National Academy of Sciences of the United States of America
Each year influenza virus infections cause hundreds of thousands of deaths worldwide and a significant level of morbidity with major economic burden. At the present time, vaccination with inactivated virus vaccine produced from embryonated chicken eggs is the most prevalent method to prevent the infections. However, current influenza vaccines are only effective against closely matched circulating strains and must be updated and administered yearly. Therefore, generating a vaccine that can provide broad protection is greatly needed for influenza vaccine development. We have previously shown that vaccination of the major surface glycoprotein hemagglutinin (HA) of influenza virus with a single -acetylglucosamine at each of the N-glycosylation sites [monoglycosylated HA (HA)] can elicit better cross-protection compared with the fully glycosylated HA (HA). In the current study, we produced monoglycosylated inactivated split H1N1 virus vaccine from chicken eggs by the N-glycosylation process inhibitor kifunensine and the endoglycosidase Endo H, and intramuscularly immunized mice to examine its efficacy. Compared with vaccination of the traditional influenza vaccine with complex glycosylations from eggs, the monoglycosylated split virus vaccine provided better cross-strain protection against a lethal dose of virus challenge in mice. The enhanced antibody responses induced by the monoglycosylated vaccine immunization include higher neutralization activity, higher hemagglutination inhibition, and more HA stem selectivity, as well as, interestingly, higher antibody-dependent cellular cytotoxicity. This study provides a simple and practical procedure to enhance the cross-strain protection of influenza vaccine by removing the outer part of glycans from the virus surface through modifications of the current egg-based process.
10.1073/pnas.1819197116
Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections.
Chen Juine-Ruey,Yu Yueh-Hsiang,Tseng Yung-Chieh,Chiang Wan-Ling,Chiang Ming-Feng,Ko Yi-An,Chiu Yi-Kai,Ma Hsiu-Hua,Wu Chung-Yi,Jan Jia-Tsrong,Lin Kuo-I,Ma Che,Wong Chi-Huey
Proceedings of the National Academy of Sciences of the United States of America
The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HAmg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HAmg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HAfg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HAmg vaccine activated more diverse B-cell repertoires than the HAfg vaccine and produced antibodies with cross-strain binding ability. In summary, the HAmg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.
10.1073/pnas.1323954111