key: cord-0003645-yhh3ydok authors: Karch, Christopher P; Matyas, Gary R; Burkhard, Peter; Beck, Zoltan title: Glycosylation of the HIV-1 Env V1V2 loop to form a native-like structure may not be essential with a nanoparticle vaccine date: 2019-01-10 journal: Future Virol DOI: 10.2217/fvl-2018-0174 sha: 01297dffaf94c1314ca46088f7b829b8383c2f73 doc_id: 3645 cord_uid: yhh3ydok nan The only surface protein of human immunodeficiency virus-1 (HIV-1) is the Env fusion protein. Env is heavily glycosylated, with glycans composing up to half of its mass [19] . During maturation of Env, the full-length gp160 is cleaved by a protease into gp41 and gp120, which remain associated forming Env heterodimers. Three heterodimers form a trimeric spike, and approximately 14 spikes decorate the surface of the virus [20, 21] . The majority, about 62-79% of the gp120 glycans present on virus, are underprocessed N-linked high mannose, because the high density of the glycosylation sites can prevent the further processing steps of the glycans by the enzymes in the Golgi apparatus [22] . Changes in the glycosylation pattern of gp120 can alter the receptor binding and infection by the virus, as well as the susceptibility to antibodies [23, 24] . In addition, the high mutation rate of HIV-1 results in an inconsistent level of glycosylation. For example, transmitter/founder viruses generally have a lower number of N-linked glycosylation sites than chronic viruses and appear to be more infectious [25] . A major effort has been undertaken to understand the role of glycans in respect to broadly neutralizing antibodies (bnAbs) to the Env protein of HIV-1 [26] . Some of these antibodies directly interact with glycans. A well-known bnAb that exclusively recognizes the glycosylation pattern is 2G12 [23] . Most of the bnAbs interacting with both the glycan and the amino acid sequence of gp120 have structural features, such as long CDRH3 loops, to penetrate the glycan shield [27] . Binding to the virus and neutralization by many of these bnAbs primarily depend on the quaternary structure of the protein, which can, however, be affected by the presence of the glycans. Between 10 and 30% of people infected with HIV-1 develop bnAbs with cross-clade neutralizing activity within 3 years of infection but while controlling the infection, it does not result in sterilizing immunity [28] . Considering that only a small fraction of antibodies to HIV-1 elicit broadly neutralizing function, the low concentration of these bnAbs in vivo, and their late occurrence during disease progression; a potentially different approach to generate bnAbs for an effective prophylactic vaccine may be necessary [26] . It raises the question: is it possible to induce high titer bnAbs with high binding affinity to the virus with an Env immunogen that is not glycosylated, but maintains the conformation of the protein? One of the most important regions of the HIV-1 Env protein is the V1V2 loop. IgG antibodies raised against this loop were inversely correlated with the risk of infection in the RV144 clinical trial [29] . Studies indicate that the V1V2 loop of the protein does not assume a unique defined structure [30] . As a result of the high density of glycans on the Env protein, most efforts in subunit vaccine development have used mammalian cells for bio-production. However, similar to the influenza hemagglutinin, a potential HIV-1 V1V2 vaccine candidate could be developed with an antigen that is expressed in E. coli, without glycosylation but with native-like conformation to induce high titers of high affinity antibodies that could prevent the initial infection [31] . The use of a carrier may be essential to the development of a successful V1V2 vaccine candidate. One such technology is the self-assembling protein nanoparticle (SAPNs). SAPNs are built on coiled-coil protein folding domains, which are composed of two or more α-helices. Glycosylation of the HIV-1 Env V1V2 loop to form a native-like structure may not be essential with a nanoparticle vaccine [32] . By choosing the correct combination of coiled-coil domains, protein folding can be optimized and the antigen of interest can be folded into a native-like conformation. This technology has been successfully applied in the development of influenza, SARS, toxoplasma and malaria vaccine candidates, in all of which the immunogens were unglycosylated [9, [33] [34] [35] [36] . Although the importance of glycosylation for viral maturation and folding of envelope proteins is clear, it remains unclear if proper glycosylation or some level of glycosylation of the immunogen is necessary for inducing protective immune response by an HIV-1 vaccine. Some attempts have been carried out to correlate the glycosylation status of the HIV-1 antigen and the immune response [37] [38] [39] . It is hypothesized that for protective immune response to HIV-1, generation of conformational antibodies is required. These antibodies can be generated only by properly folded immunogens that have either the native glycosylation pattern or are displayed on a nanoparticle carrier to enable the native-like trimeric antigen conformation. Producing subunit vaccine candidates in mammalian cells to generate glycosylation is difficult; the yield tends to be lower, and cost tends to be higher. The glycosylation pattern obtained in expression systems may be different than those present on the virion, ultimately affecting the structure of the antigen [40] . By using the SAPN technology, we were able to produce a vaccine candidate in E. coli capable of raising high titers of antibodies to the V1V2 loop. The structure of the antigen was determined to be native-like by the binding to known conformational V1V2 antibodies such as PG9, PG16 and PGT145 [31] . We hypothesize that vaccination with unglycosylated HIV-1 Env V1V2 antigens that fold into native-like confirmation may generate functional antibodies at higher titers with higher affinity, ideally leading to a better vaccine candidate. To validate this hypothesis, further studies are required to compare the functionality of antibodies from animals vaccinated with unglycosylated and glycosylated SAPN immunogens. A prime boost strategy with a vaccination using unglycosylated immunogen followed by a glycosylated immunogen can be used in an attempt to generate high titers of anti-V1V2 antibodies that recognize the virion, regardless of the glycosylation status. P Burkhard has an interest in the company Alpha-O Peptides that has patents or patents pending on the technology. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. 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