AIDS Reviews. 2019;21 Contents available at PubMed www.aidsreviews.com PERMANYER AIDS Rev. 2019;21:218-232 www.permanyer.com  f the publisher.   © Permanyer 2019 Designing Chimeric Virus-like Particle-based Vaccines for Human Papillomavirus and HIV: Lessons Learned Yoshiki Eto1, Narcís Saubi1, Pau Ferrer2, and Joan Joseph1* 1 Vall d’Hebron Research Institute, Hospital Universitari Vall d’Hebron; 2Department of Chemical, Biological, and Environmental Engineering, School of Engineering, Autonomous University of Barcelona, Barcelona, Spain Abstract Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not contain any No part of this publication may be reproduced or photocopying without the prior written permission o genetic material so that they are not infectious. VLPs maintain the same antigenic conformation to the original virus, and they could be a better vaccine candidate than live-attenuated and inactivated vaccines. In addition, compared to other subunit vaccines such as soluble protein, VLPs can stimulate both innate and adaptive immune responses effectively and safely against several pathogens by the closer morphol- ogy to its native virus. They have already been licensed as vaccines against Hepatitis B virus, human pap- illomavirus (HPV), and several veterinary diseases. Moreover, it has been investigated to prevent other viral infections including HIV. While HIV VLP-based vaccines have been studied over 35 years, none of them has been successful enough to reach even Phase III clinical trials. In this review, we summarize: (i) general features of VLPs; (ii) epidemiological data and current status of vaccine research and development on HPV and HIV; and (iii) previous studies held on HPV VLPs, HIV VLPs, and chimeric HPV/HIV VLPs including pro- duction methods and different animal immunization assays. Furthermore, we review present state of human clinical trials with VLPs and consider the potential to develop a successful preventive HIV vaccine using HPV VLP models. Finally, we discuss the benefits, limitations, and challenges of developing chimeric VLP- based HPV/HIV vaccines with recent findings, critical issues to improve VLP-based vaccines, and hot topics for the next 5 years to join the global effort to fight against these two pathogens. (AIDS Rev. 2019;21:218-232) Corresponding author: Joan Joseph, jjoseph@vhebron.net Key words Human papillomavirus. HIV. Chimeric. Vaccine. Virus-like particle. Introduction and still regarded as the most powerful means to fight against pathogens. After several years from the ap- Since the discovery of the world’s first vaccine by pearance of the classical vaccines, in other words, Edward Jenner until the present, vaccination has been live-attenuated and inactivated vaccines, progress in controlling and preventing many infectious diseases genetic science allowed us to develop subunit vaccines Correspondence to: *Joan Joseph HIV Vaccine Research Hospital Universitari Vall d’Hebron Vall d’Hebron Barcelona Hospital Campus Received in original form: 19-09-2019 Barcelona, Spain Accepted in final form: 05-11-2019 E-mail: jjoseph@vhebron.net DOI: 10.24875/AIDSRev.19000114 218 Eto, et al.: Chimeric VLP-based vaccines for HPV/HIV (SUVs). However, SUVs generally were less roughly 36.9 million people were living with HIV in immunogenic than those classical vaccines and more 2017, and 1.8 million new infections were diagnosed7. administrations were needed to reach sufficient im- Even though there is no effective preventive vaccine  f the publisher.   © Permanyer 2019 munity. The advance of SUV technology led to generate against HIV or therapeutic vaccine against AIDS yet virus-like particles (VLPs) with a higher immunogenic after more than 35 years of our vigorous investigations, effect than previous SUV models. VLPs have very sim- HIV VLP has been considered as a promising vaccine ilar appearance to the original virus and are composed candidate for its higher immunogenicity and safety of only virus structural proteins, which can be dis- compared to conventional vaccine models. Several played in a highly repetitive manner (Fig. 1)1-3. There- candidates of HIV VLPs have already been produced fore, VLPs are capable to effectively cross-link spe- and their results in animal models have demonstrated cific receptors on B cells and induce robust humoral that VLP-based HIV vaccines can elicit a high level of responses without adjuvants4. Since they do not con- HIV-1-specific neutralizing antibodies and CTL immune tain any viral genetic material inside, they lack capac- responses8. ity to replicate and are non-virulent, and composing Since both HPV and HIV are important public health proteins can form VLPs by self-assemble manner. VLPs issues in developing and industrialized countries, and No part of this publication may be reproduced or photocopying without the prior written permission o are classified as enveloped or non-enveloped the available prevention strategies have not been able (Fig. 1B)1. Non-enveloped VLPs are categorized as to stop the epidemics, a safe, effective, and affordable single or multilayered capsid protein while enveloped vaccine is immediately needed against both viruses. VLPs generally consist of glycoproteins embedded in Considering that one of the mutual main access routes the lipid membrane layers (Fig. 1B). In addition, VLPs to human body for those pathogens is the mucosal have ideal sizes for the favorable intake by dendritic genital tract, the development of a combined vaccine cells (DCs) through micropinocytosis and endocytosis, which would protect against HPV and HIV infections is resulting in activating both innate and adaptive a logical effort in the fight against these two major immunity5. They have been used as Food and Drug global pathogens. Administration (FDA) – and European Medical Agency VLP technology allows to combine multiple compo- (EMA) – approved protein SUVs against hepatitis B nents of different viruses into a single VLP. Until today, virus and human papillomavirus (HPV) (Table 1)1. VLPs several articles have been published regarding bovine can be mass produced in cell cultures such as yeast, papillomavirus (BPV)/HIV and HPV/HIV VLP-based insect, and eukaryotic cell lines. Other large-scale pro- bivalent vaccines, but those results are rather contro- tein expression systems as plant production or insect versial and none of them elicited robust HPV- and HIV- larvae have also been considered. specific humoral and T cell-mediated immune respons- HPV and HIV are both sexually transmitted viral es that could justify moving to Phase I clinical trials9-17. pathogens and are leading causes of death for hu- Thus, this review will discuss: (i) the current status, mans. In 2018, HPV infection was one of the causing limitations, and challenges of VLP-based HPV, HIV, and agents of cervical cancer, which is the second most chimeric HPV/HIV vaccine development; (ii) what has common cancer among women living in less devel- been learned in previous VLP research; and (iii) what oped areas with an approximated 570,000 new cases should be taken into account to develop chimeric HPV/ (84% of the global new cases) and an estimated HIV VLPs as an effective dual vaccine, especially fo- 311,000 deaths, of which more than 85% occurred in cused on yeast, baculovirus/insect cell, and eukaryotic low- and middle-income nations6. HPV infection is also expression systems. involved in oral and anal cancers6. While three HPV vaccines are already licensed and all of them based VLP-based HPV vaccine on VLP, they cover only major HPV genotypes (types 16, 18, 31, 33, 35, 52, and 58 are related to cervical Current vaccines and how to improve them cancer, and types 6 and 11 are related to genital warts) and still miss other less prevalent ones. Moreover, they Heretofore, three HPV vaccines have been licensed, are still prohibitively expensive for the majority of wom- and the main component of all of them is a protein en in developing countries and cervical cancer caused subunit of HPV, capsid protein L1, which self-assem- by HPV is a lasting menace in those regions. bles to form VLPs. These three vaccines are multivalent. The HIV infection causes AIDS. According to UN- Gardasil® (2006, Merck & Co.), Cervarix® (2009, Glaxo- AIDS’s 2018 fact sheet, it has been estimated that SmithKline), and Gardasil®9 (2014, Merck & Co.) are 219 220 a) a b c C AIDS Reviews. 2019;21 b) c) Figure 1. A: Schematic representation of structural characteristics of viral particles and virus‐like particles (VLPs). (a) Comparison of external characteristics. (b) Cross‐section representation showing internal distinctions. (c) Transmission electron microscopy images of influenza viral particles and plant‐produced influenza VLPs2; B: Structural diversity of VLPs. Both capsid-based and enveloped VLPs can be single or multilayered and composed of single or multiple proteins. Single layered non-enveloped VLPs can be assembled from (i) a single protein (e.g., hepatitis B core antigen [Clarke et al., 1987; Whitacre et al., 2009]); or (ii) two proteins (e.g., cowpea mosaic virus VLPs [Saunders et al., 2009]). Double layered non-enveloped VLPs can be assembled from (iii) two proteins (e.g., pap- illomavirus L1 and L2 VLPs [Kirnbauer et al., 1993]); or (iv) four proteins (e.g., Foot-and-mouth disease virus [Porta et al., 2013]). Triple-layered VLPs (v) have been assembled from four coat proteins of bluetongue virus (Hewat et al., 1994) and rotavirus (Conner et al., 1996). For enveloped VLPs, expression of one (vi) or two glycoproteins (vii) will form a single layer, as demonstrated by the expres- sion of influenza virus hemagglutinin (D’Aoust et al., 2008) and the coexpression of both hemagglutinin and neuraminidase (Chen et al., 2007), respectively. Double layered enveloped VLPs can be formed by the (viii) inclusion of structural proteins (e.g., influenza VLPs [Latham and Galarza, 2001]) and may possess (ix) multiple glycoproteins on their surface (Latham and Galarza, 2001; Pushko et al., 2011)1; C: structure of VLPs for a range of viruses. Capsid morphology and the proteins expressed to achieve this are depicted for each virus. In each case, the electron micrograph shows the VLP produced. Abbreviations: BTV, bluetongue virus; HCV, hepatitis C virus; HEV, hepatitis E virus; HPV, human papillomavirus 16; SIV – HIV, hybrid VLP between simian immunodeficiency virus gag and human immunodeficiency virus Env3. No part of this publication may be reproduced or photocopying without the prior written permission o  f the publisher.   © Permanyer 2019 Eto, et al.: Chimeric VLP-based vaccines for HPV/HIV Table 1. Compositions of commercial VLP‑based vaccines (adapted from Lua et al., 2013)1 Vaccine Composition per dose  f the publisher.   © Permanyer 2019 Protein Adjuvant Others Engerix‑B hepatitis HBsAg (20 μg) Aluminum hydroxide (500 μg) 2‑Phenoxyethanol (5.0 mg) B vaccine (1 mL/dose) Recombivax HB hepatitis HBsAg (10 μg) Aluminum Sodium chloride (9 mg) B vaccine (1 mL/dose) hydroxyphosphate (500 μg) Sodium borate (70 μg) Gardasil human HPV 6 L1 (20 μg) Aluminum hydroxyphosphate Sodium chloride (9.56 mg) papillomavirus vaccine HPV 11 L1 (40 μg) sulfate (225 μg) L‑histidine (0.78 mg) (0.5 mL/dose) HPV 16 L1 (40 μg) Polysorbate 80 (50 μg) HPV 18 L1 (20 μg) Sodium borate (35 μg) Cervarix human HPV 16 L1 (20 μg) AS04 adjuvant system (50 μg of Sodium chloride (4.4 mg) papillomavirus vaccine HPV 18 L1 (20 μg) 3‑O‑desacyl‑40 monophosphoryl Sodium dihydrogen (0.5 mL/dose) lipid A, 500 μg aluminum phosphate No part of this publication may be reproduced or photocopying without the prior written permission o hydroxyphosphate) dehydrate (0.624 mg) Hecolin hepatitis E vaccine HE antigen (30 μg) Aluminum hydroxide (800 μg) Buffered saline (0.5 mL/dose) Gardasil9 human HPV 6 L1 (30 μg) Aluminum hydroxyphosphate Sodium chloride (9.56 mg) papillomavirus vaccine HPV 11 L1 (40 μg) sulfate (500 μg) L‑histidine (0.78 mg) (0.5 mL/dose) HPV 16 L1 (60 μg) Polysorbate 80 (50 μg) HPV 18 L1 (40 μg) Sodium borate (35 μg) HPV 31 L1 (20 μg) HPV 33 L1 (20 μg) HPV 45 L1 (20 μg) HPV 52 L1 (20 μg) HPV 58 L1 (20 μg) HBsAg: hepatitis B virus surface antigen; HPV: human papillomavirus. quadrivalent (genotypes 6, 11, 16, and 18), bivalent that even one dose of bivalent vaccine would have (genotypes 16 and 18), and 9-valent (genotypes 6, 11, merit for prophylactic motives21. Besides Gardasil®9, 16, 18, 31, 33, 45, 52, and 58), respectively (Fig. 2)18. second-generation of VLP-based preventive HPV vac- The tetravalent Gardasil® has been replaced by non- cines has been developed and tested preclinically. avalent Gardasil®9 so that Cervarix® and Gardasil®9 are Regarding the production system, different species of currently used to prevent HPV infections. While HPV yeast such as Pichia pastoris22 and Hansenula polymor- genotypes 6 and 11 cause 90% of genital warts19, gen- pha23 have potential to produce L1 protein with lower otypes 16 and 18, 31, 33, 45, 52, and 58 and some cost and higher yields compared to Saccharomyces others cause cervical, vulvar, vaginal, and anal cancer, cerevisiae used for Gardasil® and Gardasil®9, or com- and genotypes 16 and 18 are responsible for nearly pared to the baculovirus/insect cell (Hi-5 Rix4446 cells 70% of all the cervical cancer cases6. Although HPV derived from the insect Trichoplusia ni) expression sys- vaccination has been effective and reduced HPV infec- tem used for Cervarix®. Based on the fact that the an- tions significantly among the vaccinees, completing all tibodies against minor papillomavirus (PV) capsid pro- the immunization schedules of two or three doses are tein L2 have a cross-neutralizing activity, monovalent economically challenging even in high-income coun- L1 expressing RG1 epitope (aa17-36 of capsid protein tries, and the HPV vaccines are still very expensive and L2) might work to protect from other HPV genotype not affordable in low-income countries where most infections19. Moreover, Novartis Vaccines and Diagnos- HPV-related cancers occur, and the vaccination cover- tics (Emeryville, CA, USA) stated a method to generate age is very low20. From these results, a cheaper and yeast-expressed mosaic VLPs composing both HPV-6 stable HPV vaccine ideally potent enough with one and -16 L1 proteins24. Furthermore, as vehicles of edi- dose is urgently needed. In fact, preliminary data show ble HPV vaccines, Asahi Glass (Kanazawa, Japan) pat- 221 AIDS Reviews. 2019;21  f the publisher.   © Permanyer 2019 a b f c d g No part of this publication may be reproduced or photocopying without the prior written permission o e h Figure 2. Schematic diagram to depict the next generation of preventive human papillomavirus (HPV) vaccines based on HPV capsid proteins (L1 and/or L2)18. A: Cervarix composed of HPV-16 and HPV-18 virus-like particles (VLPs); B: Gardasil composed of HPV-6, HPV- 11, HPV-16, and HPV-18 VLP; C: Multivalent VLP vaccines composed of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 VLPs (V503 Merck); D: L1 capsomer vaccine; E: Chimeric L1-L2 VLP vaccine with L2 on the surface; F: L2 peptide vaccine; G: Concatenated L2 peptide vaccine; H: L2 peptides displayed on the surface of bacteriophage VLP. ented a recombinant yeast Schizosaccharomyces morpha23, were used to produce several types of HPV pombe expressing HPV-16 L1 protein, and Genome, VLPs. Furthermore, the VLPs were generated by bacu- Inc. (Pohang, Korea) patented transgenic plants ex- lovirus in Sf9, Sf21, Hi5 insect cells26-28, and Leishma- pressing recombinant HPV L124. These edible vaccines nia29. The antigenic conformation of HPV is most ac- were developed with the intention of lowering the cost curately retained by mammalian cells followed by of HPV vaccine and improve its availability regardless baculovirus/insect, plant, yeast, and E. coli protein of regions and poverty. Ultimately, an ideal preventive expression systems24. In addition, the codon-optimiza- HPV vaccine should protect vaccinees from all 15 of tion for host cells plays an important role to improve the high-risk HPV genotypes24. the yield of HPV L1 protein30. Although there are differ- ences in heterologous protein productivity, regardless HPV VLP production systems: yeast, of expression systems, appropriate HPV L1 proteins baculovirus, and other eukaryotic cells were generated, and the VLPs were self-assembled properly. Furthermore, since 1998, the disassembly HPV VLPs have been produced previously using dif- and reassembly of HPV VLPs by exchanging buffers ferent expression systems such as Escherichia coli, have been considered as an important process to im- yeast, baculovirus/insect cells, and plants. Among prove the quality and uniformity of VLP because the L1 yeast strains, S. cerevisiae25, P. pastoris22, and H. poly- proteins could stick together improperly during the pu- 222 Eto, et al.: Chimeric VLP-based vaccines for HPV/HIV rification process31. In addition, the new expression other SUVs. As HIV-1 polyprotein precursor can form system, Leishmania, has a great potential to produce 100 nm to 120 nm VLP, Gag-based and Env-based VLPs massively, 2-3 mg/500 mL media29, which is VLPs were studied separately first39,40, and then chi-  f the publisher.   © Permanyer 2019 roughly 10 times of amount compared to one of bacu- meric Env-Gag HIV VLPs have been targeted as a lovirus/insect cell systems generating 0.25 mg of VLP vaccine model41 to improve immunogenicity. Further- from 0.5 L of insect cells in 199326. more, chimeric VLPs of HIV and other viruses such as influenza virus, Bovine/HPV (B/HPV), and hepatitis E HPV VLP immunogenicity virus have also been studied9-17,42,43. Nowadays, for the sake of enhancing VLP-derived immune responses, All immunogenicity studies have been focused on especially T-cell mediated immunity, delivering VLPs neutralizing antibodies rather than T-cell mediated im- with different adjuvants such as toll-like receptors have munity because the neutralizing antibodies have been been assessed, and it is known that nonmethylated CG shown to have the main role in the prevention of HPV motifs could generate higher T-cell mediated cytotoxic- infection as considered in the commercial vaccines32,33. ity when they were codelivered with VLPs44. Moreover, Interestingly, both Gardasil® (genotypes 6, 11, 16, recent studies suggest that the exposure of highly con- No part of this publication may be reproduced or photocopying without the prior written permission o and 18) and Cervarix® (genotypes 16 and 18) showed served epitopes of HIV Env by removing glycosylation cross-protection against HPV-31 and HPV-45 due to sites could improve the production of broadly neutral- their similarity to HPV-16 and HPV-184,34. This suggests izing antibodies (bNAbs) by HIV Env-based VLPs8. In that Gardasil® 9 and the next generation of HPV VLP addition, natural killer cell immune responses were vaccines should be evaluated if they can reduce the mediated by Gag-VLP through activating and maturat- incidence of infection with other HPV genotypes be- ing DCs45. Furthermore, although DC-based immuniza- sides the targeted ones by cross-protection. tion is still intricate, DCs loaded with HIV-1 VLP was Recently, in 2017, Huber et al.35 created HPV L1-L2- suggested to be used as a supplementary boost regi- based VLP targeting cutaneous HPV. Minor capsid men for HIV-1 VLP vaccination because DCs are po- protein L2 was considered to extend the genotype- tent and indispensable antigen-presenting cells to in- restricted protection generated by the current HPV L1- duce HIV-1 specific humoral immunity8. based vaccine. It showed not only humoral immunity What is more, a few independent non-human primate against HPV genotypes included in the VLPs, also had immunization studies with SIV Gag VLPs demonstrated cross protections against other HPV genotypes, and cross-protection between SIV Env and Gag (re- this could be a promising next-generation HPV vaccine viewed in8). All the research groups detected elevated candidate. Env-specific or neutralizing antibody responses after In favor of facilitating vaccine administration, SIV or SHIV challenge in rhesus macaques which were non-needle injection routes such as nasal and oral immunized with only SIV Gag VLPs. administration should be considered. In addition, further Another series of studies in small animal models dem- studies of dose-route responses should be performed onstrated that membrane-anchored flagellin and CD40 li- and compared with three-dose immunization schedule. gand have been shown as effective adjuvants to enhance HIV-1-specific immune responses after incorporating vari- VLP-based HIV vaccine ous adjuvants into HIV-1 VLPs (reviewed by Zhao et al8). While bNAbs against HIV are considered as a crucial Current status and its challenge factor to prevent HIV infection, it does not seem suffi- cient and the induction of HIV-specific T-cell-mediated Although VLP-based HIV vaccine studies have been immune responses is also essential to develop a pro- held over 30 years, none of the HIV VLP vaccine can- phylactic vaccine against HIV. In other words, an op- didates has shown to be protective enough. A few of timal HIV vaccine should induce innate mucosal, hu- them have successfully reached clinical trials but were moral, and cellular immunity specific for HIV. Another not assessed in efficacy trials due to low immunogenic- difficulty in developing preventive HIV vaccines is ity and safety properties36-38. However, several HIV’s high mutation rates and genetic diversity so that important factors have been found regarding its im- designing a universal and cross-clade HIV vaccine is munogenicity in comparison to subunit proteins. In extremely challenging. Therefore, several researchers general, HIV VLPs could induce stronger humoral and have been currently aiming to select and target more cellular immunity than HIV recombinant proteins or conserved regions/epitopes to their HIV VLP mod- 223 AIDS Reviews. 2019;21 els8,46. Moreover, finding perfect animal models to HIV VLP immunogenicity studies evaluate protective effect of vaccine candidates is one of other big issues. Evident immunogenicity data have Tsunetsugu-Yokota et al.57 separated DCs and T cells  f the publisher.   © Permanyer 2019 been described by many authors after non-human pri- from fresh peripheral blood mononuclear cells of mate immunization with several HIV-1 vaccine candi- non-infected and HIV-infected individuals and observed dates47, but these had disappointing results in hu- that pulsing DCs with 1 or 10 µg/mL HIV-1 Gag p55 mans48. Nowadays, the humanized mouse model has VLP for 2 days induced perforin expression in Gag- also been considered as an alternative animal model specific CD8 T cells. Furthermore, BALB/c mice were to assess immunogenicity and protection as it can inoculated intradermally with 20 µg of HIV-1 Gag p55 mimic the human immune system49. Furthermore, the VLPs twice at 3-week interval, and statistically signifi- conformational changes and glycan shield of the HIV cant anti-Gag humoral responses were detected58. envelope are other challenges for the development of Immunogenicity data with three types of HIV-1 Env- an effective HIV-1 vaccine50,51. Finally, understanding based VLPs were demonstrated by Crooks et al.59 the immune correlates of protection against HIV-1 Guinea pigs were immunized with (i) “naked VLPs”, would be an important key to develop an efficacious which do not bear Env, (ii) “SOS-VLPs” bearing disul- No part of this publication may be reproduced or photocopying without the prior written permission o HIV-1 vaccine52. fide-shackled functional trimers, and (iii) “UNC-VLPs” that present uncleaved nonfunctional Env or soluble HIV VLP production in yeast, baculovirus, monomeric gp120 3 times on days 0, 43, and 97 by a and other eukaryotic cells combination of intradermal and intramuscular (i.m.) routes in two locations each. UNC- and SOS-VLPs im- HIV VLPs have been also generated in several ex- mediately induced anti-gp120 antibodies while in na- pression systems. Yeast expression system was used ked VLP case, humoral immunity was barely detected to produce HIV VLPs for the 1st time in 1987 by Adams after third dose. Ultimately, low level of neutralizing et al. using the yeast retrotransposon, Ty, that encodes activity was found in all candidates. a set of proteins that are assembled into VLPs, Ty- Tagliamonte et al.60 administered 20 µg of HIV Gag VLPs. They showed that the major structural compo- p55-based VLPs presenting trimeric HIV-1 gp140 spikes nents of Ty-VLPs were proteolytic products of the pri- in BALB/c mice subcutaneously twice at the 3-week mary translation product, p1, and such protein p1 interval and acquired statistically significant anti-gp140 alone can form Ty-VLPs by itself. Moreover, they dem- activity while anti-Gag activity was not analyzed. onstrated that p1 fusion proteins, consisting of most of Benen et al.46 assessed HIV Gag VLP-based immu- p1 and part of HIV-1 gp120 could form hybrid HIV: Ty- nogen presenting membrane proximal external region VLPs53. In 2002, Sakuragi et al. produced HIV-1 p55 (MPER) of HIV-1 gp41. They demonstrated in an im- (Gag) VLPs by budding from yeast spheroplasts, in munization study in rabbits that priming with DNA and other words, yeast cells without cell wall54. However, boosting with VLPs generated that low titers of anti- to get spheroplasts, first, the cell walls needed to be MPER antibodies and low neutralizing activity. gently enzymatically digested by Zymolyase-100T be- Poteet et al.61 inoculated HIV-1 Gag/Env VLPs with cause thick yeast cell walls were considered as one monophosphoryl lipid A (MPLA) adjuvant to C57BL/6 difficulty to secret proper HIV VLP54. Baculovirus/insect mice through different routes including a novel oral, cell system was mainly chosen to produce HIV VLPs buccal cheek subcutaneous administration. After trying in 1990s and 2000s, especially because of higher several combinations of injection routes, they conclud- yields compared to yeast system40,55. Recently, human ed that an intranasal prime sub-cheek boost regimen embryonic kidney cells (HEK) 293 cells have relatively of HIV-1 Gag/Env VLPs with MPLA adjuvant has a more efficient protein production than baculovirus/in- strong potential to induce Env-specific Th1-oriented sect cell expression system and it has been the main HIV-specific immune responses. expression system of HIV VLP production. HEK 293 Vzorov et al.62 specifically modified the transmem- cells have been adapted and can produce 1 mg/L of brane spanning (TMS) and cytoplasmic tail (CT) do- VLP or more in a few days and can produce gag-env mains of HIV-1 Env. They demonstrated that the im- VLPs. For example, Cervera et al. showed HIV-1 VLP munization of guinea pigs by a construct containing a production of 2.8 µg of recombinant Gag-GFP/ml of short version of the TMS domain induced the highest HEK 293 cell culture56. titers of anti-Env IgG immune responses. In addition, 224 Table 2. Chimeric B/HPV‑S/HIV VLP production and immunogenicity in small animal and non‑human primate models Year Expression Cells Recombinant Animal model Route Dose Schedule Immunogenicity References system proteins 1998 Baculovirus/ Sf‑9 cells L1 (BPV‑1) + BALB/c mice SC 20 μg 0, 2, 4 weeks Anti‑BPV1 VLP Peng et al.14 insect cells P18‑I10(HIV‑1) Ab and CTL 1998 Baculovirus/ Sf‑9 cells L1 (BPV‑1) + C57BL/6J mice IN, IM 50 μg × 2 Day 0, 21 IgG, IgA, CTL Liu et al.15 insect cells P18‑I10(HIV‑1) 1999 Baculovirus/ Sf‑9 cells L1 (BPV‑1) + C57BL/6 mice ID 10 μg × 3 0, 2, 4 weeks IgG, anti‑CCR5 Chackerian insect cells mCCR5 Ab, chemokine et al.17 2000 Baculovirus/ Sf‑9 cells L1 (BPV‑1) + BALB/c mice IM 20 μg × 2 Day 0, 21 IgG, CTL Liu et al.16 insect cells P18‑I10 (HIV‑1) + C57BL/6J mice RT (HIV‑1) + HLA‑A2.1/Kb Nef (HIV‑1) transgenic (H‑2b) mice 2002 Baculovirus/ Sf‑9 cells L1 (BPV‑1) + V3/ BALB/c mice IM, IVA, IR 50 μg × 2 Day 0, 14 IgG, IgA, CTL Liu et al.9 insect cells P18‑I10 (HIV‑1) 2002 Baculovirus/ Sf‑9 cells L1 (HPV6b) + P27 Pigtailed IM, IR IM 20 μg × 8, 11, 14 weeks AntiHPV L1 ab, Dale et al.10 insect cells gag (SIV)/ macaques 7 as prime IFN‑Ƴ, SHIV tat (HIV‑1)/ IR 20 μg × challenge rev (HIV‑1) 3 as boost 2004 Baculovirus/ Sf‑9 cells L1 (BPV‑1) + C57BL/6 mice, Mice: SC, mice: 5 μg Mice: 0, 2, IgG, Challenge Chackerian insect cells ptCCR5 pig‑tailed Macaques: ×3 4 weeks with S/HIV et al.11 macaques IM macaques: Macaques: 20‑25 μg × 9 times 9 during>2 years 2009 293 GPR 293 GPR HIV‑1 Nef+HPV16 C57BL/6 mice SC 10 μg × 3 0, 2, 4 weeks IFN‑Ƴ Di Bonito HIV‑1 HIV‑1 E7 et al.12 inducible inducible packaging packaging cells cells 2013 Baculovirus/ Sf‑9 cells L1 (BPV‑1) + BALB/c mice Oral 10 μg × 3 0, 2, 4 weeks mAb, IgG, IgA, Zhai et al.13 insect cells gp41 (HIV‑1) HIV neutralizing assay SC: subcutaneous, ID: intradermal, IM: intramuscular, IN: intranasal, IP: intraperitoneal, BPV: bovine papillomavirus, HPV: human papillomavirus, SIV: simian immunodeficiency virus. Eto, et al.: Chimeric VLP-based vaccines for HPV/HIV 225 No part of this publication may be reproduced or photocopying without the prior written permission o  f the publisher.   © Permanyer 2019 AIDS Reviews. 2019;21  f the publisher.   © Permanyer 2019 No part of this publication may be reproduced or photocopying without the prior written permission o Figure 3. Schematic representation of chimeric B/HPV and S/HIV-1 proteins for virus-like particles (VLP)-based vaccine development (adapted from Sadeyen et al.64). A: Wild type model of B/HPV L1 protein. B-M: The insertion point of HIV protein in B/HPV L1 protein is indicated. 360 copies of each protein form a VLP. (i) BPV L1 VLP-based chimeric HPV/HIV model. B and C: HIV protein was inserted at C-terminal. D,H,J: D-E loop 130-136 of BPV1 L1 protein was replaced by mCCR5 or HIV-1 epitope. E: FG loop 275-285 of BPV1 L1 protein was replaced by mCCR5. F: HI loop 344-350 of BPV1 L1 protein was replaced by mCCR5. G: Purified BPV1L1 VLPs were biotinylated and EC domains were bound to those biotins. (ii) HPV L1 VLP-based chimeric HPV/HIV model. K-M: SIV or HIV protein was inserted at C-terminal. (iii) Other chimeric HPV/HIV model. N: BPV1L1, HPV16E7 B, HPV16E7 CTL, HIV1 P18-I10, HIV1 Nef CTL, and HIV RT CTL are combined as seen above. O: gag-pol-nef HIV VLPs with modification at nef c-terminal by HPV16 E7 were produced. BPV: bovine papil- lomavirus; HPV: human papillomavirus; SIV: simian immunodeficiency virus; WT: wild type. 226 Eto, et al.: Chimeric VLP-based vaccines for HPV/HIV Table 3. Ongoing clinical trials of HPV VLP and other other VLP models65 HPV VLP Estimated study  f the publisher.   © Permanyer 2019 completion date A study of V503 (a multivalent HPV L1 VLP vaccine) in preadolescents and adolescents (V503‑002) December 3, 2020 Long‑term follow‑up of broad‑spectrum HPV vaccine study in women (V503‑021) January 1, 2024 A PMS study to monitor the safety of GSK Biologicals’ HPV vaccine in female Chinese subjects December 4, 2020 Immunobridging Study of 9vHPV vaccine (V503) in Chinese females 9‑45 years of age (V503‑024) September 17, 2024 Other VLP models Estimated study completion date VRC 313: a trivalent VLP encephalitis vaccine (WEVEE) in healthy adults February 2021 Trial of a chikungunya vaccine, PXVX0317 CHIKV‑VLP, in healthy adults December 2020 No part of this publication may be reproduced or photocopying without the prior written permission o Venezuelan equine encephalitis monovalent virus‑like particle vaccine (VEEV) January 2021 Long‑term immunogenicity of the norovirus GI.1/GII.4 bivalent VLP vaccine (NoV vaccine) in adults October 26, 2021 A Phase 2 open‑label study to assess the safety and immunogenicity of an alum‑adjuvanted December 15, 2020 chikungunya virus‑like particle vaccine (PXVX0317) in prior recipients of other alphavirus vaccines (WRAIR) HPV: human papillomavirus, VLP: virus‑like particle, HPV: human papillomavirus, PMS: postmarketing surveillance, GSK: GlaxoSmithKline, 9vHPV: 9‑valent human papillomavirus. VLPs with high Env content and containing the CT by Dale et al.10 In all cases, VLPs were produced by trimerization sequence improved neutralization activity baculovirus/insect sf9 cell expression system. In 2013, and antibody avidity in guinea pig. Zhai et al.13 constructed BPV-1 L1 VLP harboring (i) B/T Beltran-Pavez et al.63 demonstrated that rabbit im- cell conserved epitopes from MPER of HIV-1 gp41 and munization with HIV-1-Gag VLPs containing 4E10-se- (ii) the linear epitopes recognized by neutralizing lected envelope variant (LR1-C1), induced humoral monoclonal antibodies 2F5 and 4B10, which were in- responses to 4E10-proximal region. serted in D-E loop of L1 protein. While all the previous Taken as a whole, HIV VLPs can be safe and im- VLPs were designed to add HIV epitopes into B/HPV munogenic using different immunization routes regard- L1-based VLPs, in 2009, the incorporation of HPV pro- less of coadministration of adjuvants and should be tein into HIV-1-based VLPs produced in HEK 293 cells continuously pursued to acquire an effective HIV vac- was first reported by Bonito et al.12 cine. Immunogenicity of chimeric HPV/HIV Chimeric VLP-based HPV/HIV vaccine VLP-based vaccines Current status and vaccine production Liu et al.9 investigated in BALB/c mice whether mu- cosal administration of chimeric BPV/HIV VLP could To the best of our knowledge, the production of elicit mucosal cellular and humoral immune responses chimeric PV/HIV VLP was first described by Peng et al. to BPV VLP and incorporated HIV epitopes. They de- in 199814, using BPV/HIV VLP to present HIV epitopes. tected specific antibodies for BPV-1 VLP and HIV-1 Afterward, Chackerian et al.17 published a research CTL epitope P18 from gp120 in serum by i.m. admin- article regarding chimeric BPV VLP with CCR5 core- istration but not intrarectal (i.r.) or intravaginal (i.va) ceptor, which is required for HIV entry (Table 2 and immunization. Regarding VLP specific IgA, it was high- Fig. 3)9-17,64. Henceforth, the production and immuno- er in the intestine by i.r. than i.va administration, and genicity data of BPV/HIV were introduced by Liu higher in vaginal by i.m. than i.r. or i.va. administration. et al.9,15 and Liu et al16 and those data of HPV/HIV VLP CTL precursor cells specific for HIV P18 were found in 227 AIDS Reviews. 2019;21 spleen from all three routes of immunization but in infected patients but it could not improve HIV-specific Peyer’s patches only from i.m. or i.r. immunization. immune responses in them. Finally, DNA and recom- Dale et al.10 designed HPV genotype 6b L1 VLPs binant modified vaccinia virus Ankara (MVA) vaccines  f the publisher.   © Permanyer 2019 incorporating SIV Gag p27 and HIV-1 tat and vaccinated expressing HIV-1 VLP were assessed in Phase I trial pigtailed macaques with DNA encoding SIV gag, HIV- for safety and immunogenicity38. The MVA vaccine pro- 1 tat, and HIV-1 rev or HPV/SHIV VLP prime intramus- ducing HIV-1 VLPs of HIV-1 Gag, PR, RT, and Env was cularly and with three VLP boosters intrarectally, com- well tolerated and elicited different patterns of T cell paring DNA prime/HPV VLP boost regimen versus all and Ab responses when administered alone or in com- HPV/SHIV VLPs. However, they could detect only weak bination with the DNA vaccine producing HIV-1 VLPs antibody or T cell responses to the chimeric SHIV an- composed of HIV-1HXB-2 Gag, HIV-1BH10 PR and RT, tigen in DNA prime/HPV VLP boost regimen, but not in and Env, Tat, Rev, and Vpu derived from a recombi- the all HPV/SHIV VLP group. nant of the HXB-2 and ADA strains of HIV-1. Di Bonito et al.12 fused HPV genotype 16 E7 to Nef- Various chimeric VLP-based vaccine candidates en- mutant, inserted into HIV-1 gag-pol VLP, and vacci- tered clinical trials such as the anti-influenza A M2–HB- nated C57BL/6 mice subcutaneously 3 times over cAg VLP vaccine, the anti-HIV p17/p24:Ty VLP, two No part of this publication may be reproduced or photocopying without the prior written permission o 4 weeks at 2-week interval. The culture of the murine anti-malaria vaccines, the nicotine-Qb VLP and the splenocytes demonstrated an anti-E7 CTL activity. Fur- anti-Ang II Qb VLP, and other chimeric VLP vaccine thermore, the vaccinated mice were challenged with candidates went on preclinical trials66. tissue culture number one (TC-1) tumor cells causing Moreover, several clinical trials for other VLP-based HPV-related tumor 2 weeks after the last VLP inoculum. vaccines are on-going: Venezuelan Equine Encephalitis, The mice inoculated with the chimeric VLPs were pro- Western Equine Encephalitis, Eastern Equine Encephali- tected after tumor challenge. Furthermore, effective tis, Chikungunya virus, and Norovirus (Table 3)65. Nef-specific CTL activity was detected. BPV L1 VLPs presenting HIV-1 epitopes from MPER Expert commentary of gp41 constructed by Zhai et al.13 and were inocu- lated to BALB/c mice orally, and induced strong vagi- Chimeric VLP technique has brought us to develop nal IgG responses against BPV while only weak vaginal safer, broader, and more potent vaccine candidates HIV-specific secretory IgA responses were detected. with higher protective effect to fight against high preva- They confirmed that IgG and mucosal secretory IgA lent and emerging diseases such as malaria, dengue were elicited against 2F5 and MPER. The induced an- virus, chikungunya virus, HPV and HIV as well as to treat tibodies recognized native MPER in HIV-1 infected lifestyle diseases such as nicotine addiction and hyper- cells and were able to partially neutralize infectivity tension. Three HPV VLP vaccines are already licensed from HIV-1 viruses of clade B and C. to prevent HPV infection. Thus, the generation of chime- ric HPV/HIV vaccine holding HIV antigens would be a Human clinical trials using VLPs logical effort to fight against both pathogens. In Table 2,9-17, we summarize preclinical development In the near future, more VLP-based vaccine candi- of previous chimeric BPV/HIV or HPV/HIV VLP vaccine dates will enter human clinical trials. After the licensure candidates tested in small animal and non-human pri- of three VLP-based HPV vaccines, four clinical trials mate models, but none of the immunogenicity results are currently on-going to follow-up the safety and long- obtained in those studies were satisfactory enough to term efficacy (Table 3)65. Regarding HIV VLP, only a reach human clinical trials. One reason could be the few candidates have been tested in clinical trials. The insertion point of the HIV immunogen into HPV protein first candidate, p17/p24:Ty, where yeast-derived Ty to elicit HPV/HIV-specific immune responses. Many of protein containing 25% HIV-1 p17 and 79% HIV-1 p24, HPV/HIV chimeric designs fused the HIV epitope to the was tested in Phases I and II36. They only induced low C- terminal of HPV L1 protein. According to Sadeyen et level of HIV-specific humoral and cell-mediated im- al.64, the insertion of Hepatitis B virus epitope into the mune responses and were discontinued. Another can- loops located in HPV L1 protein could increase the im- didate, p24-VLP, which is HIV VLP composed of Gag munogenicity. Thus, the inserted epitope would be ex- p24, was reached to Phase II37. The VLPs were admin- posed outside when they form VLP. Furthermore, it istered with or without zidovudine, a nucleoside re- would be important to incorporate conserved HIV-1 epi- verse transcriptase inhibitor to asymptomatic HIV-1 topes from HIV-1 V2 loop region of the envelope into 228 Eto, et al.: Chimeric VLP-based vaccines for HPV/HIV VLPs, which recent data67 suggest is more promising to ing influenza virus, encephalitis virus, and Chikungu- induce humoral immunity against the virus. On the other nya virus will foster the advances in chimeric VLP tech- hand, we always must keep in mind that the size of HIV nology for HPV and HIV.  f the publisher.   © Permanyer 2019 epitopes inserted into HPV VLP would be an important In addition, effective adjuvants for multiple VLPs will issue to obtain the antigen conformation and VLP mor- be assessed to improve the efficacy and immunoge- phology. The inclusion of epitopes to induce bNAbs nicity. As a result, more chimeric VLP vaccines will be directed against lineal sequences such as MPER might tested in Phase I clinical trials and a few will enter in be worth considering. Besides that, as for the design of Phase II trials in the next 5 years. chimeric HIV/HPV VLPs, most research groups used These days, not only baculovirus/insect cell expres- HPV VLP as a backbone to insert HIV epitopes, but HIV sion system, and yeast expression system, but also VLPs could also be a base to add HPV epitopes as done other expression systems such as E. coli and plant by Bonito et al.12 In chimeric vaccine models, it is crucial have been investigated to produce VLPs more effec- to choose highly immunogenic antigens from interested tively. Moreover, other VLP-based influenza virus vac- pathogens and to design ideal chimera conformations cine candidates are currently tested in human clinical which can induce protective immune responses for all trials. In addition, although the immunogenicity of HPV No part of this publication may be reproduced or photocopying without the prior written permission o of them. Furthermore, it cannot be ignored that the con- L2 itself is known to be low, various amino acid regions tamination of VLPs with the residual components from of HPV-16 L2 protein hold highly conserved neutraliz- host protein-expressing cells may cause undesired ac- ing epitopes that have potential to improve the current tivation of innate and adaptive immune responses5 and HPV L1 based vaccine to prevent other HPV geno- denatured VLPs tend to induce lower immunogenicity types24,30. These new designs might improve our un- than properly formed ones68. Thus, the purity and stable derstanding and be applicable to chimeric HPV/HIV formulation of VLPs is an important factor to induce model. Furthermore, HIV-1 vaccine candidates con- expected immunity ef­ ficiently. Overall, to reduce the taining HIV envelope glycoprotein gp120 (bivalent) or cost of vaccines and improve the coverage of vaccines, gp140 (trimeric) are now being tested in human vac- scientific community is working to improve VLP-based cine efficacy trials65 and they have great potential to vaccine addressing these critical issues: (i) formulation be applied to develop chimeric HPV/HIV VLP-based level (e.g., types of adjuvants and stabilizers), (ii) ad- vaccines by incorporating them into the HPV L1 VLP. ministration route (e.g., oral, i.va or i.m.), (iii) delivery In addition, disulfide-stabilized, cleaved trimeric form vehicles (e.g., liposome, polylactide-glycolide micropar- of HIV-1 gp140, SOSIP69, which displays conforma- ticles, or alginates), and (iv) modes of delivery (e.g., nee- tional epitopes recognized by bnAbs is going to be dle, spray, or potable liquid). tested in Phase I clinical trial. For T-cell immunogens against HIV-1, mosaic immu- Five-year view and concluding remarks nogens, which were designed to provide maximum coverage of conserved regions of HIV-1, have been VLP technology will achieve great progress in pro- studied70. Another candidate, the “HIVACAT T-cell im- duction methods and efficiency, structure morphology munogen” (HTI), which was designed to cover T-cell as well as chimeric VLP designing in the next 5 years. targets, against which T-cell responses are predomi- VLPs are structurally stable and can be manipulated nantly observed in HIV-1-infected individuals with low to display heterologous protein on their surface or to HIV-1 viral loads, has also been investigated71. carry nanoparticles inside by disassembly and reas- Recently, a new concept, called virus-like-vaccines sembly process with chemical environment changes (VLVs), of merging VLPs and replication-deficient vi- that greatly broadened the possibility of VLPs as pro- rus vectors has been suggested for HIV vaccine de- phylactic and therapeutic vaccines. velopment72. VLPs can robustly yield antibody and In July 2015, a chimeric vaccine RTS,S (hybrid VLP helper T cell responses, whereas the virus vectors of P. falciparum CS protein and Hepatitis B surface can induce potent cytotoxic T cell responses. VLVs antigen) with adjuvant AS01, called RTS,S/AS01 (com- first infect cells and then produce VLPs in situ, but mercial name Mosquirix) was approved by EMA and due to the replication deficiency, the infection cannot WHO launched a pilot project for vaccination on April propagate. Therefore, VLVs can lead both humoral 23, 2019, in Malawi, planning to perform this project and cell-mediated immunity safely and effectively, also in Ghana and Kenya later in 2019. This success and this method could be applied to chimeric VLP and results of clinical trials for other VLP models includ- model. 229 AIDS Reviews. 2019;21 Classically, chimeric VLP was designed by geneti- also in Africa where any HPV VLP vaccine trial has cally inserting foreign antigen DNA sequence into an- never been conducted yet. other which was the base for VLP-forming protein, but, Therefore, a safe, affordable, effective chimeric vaccine  f the publisher.   © Permanyer 2019 nowadays, target antigens and base VLPs can be pro- against HPV and HIV which is ideally easy to deliver and duced separately, and then they could be linked using administer, require less doses, and stay stable at room either covalent or noncovalent binding73. Through this temperature would ultimately be the best solution. Even method, the exposure of target antigens on the surface if we had acquired promising preclinical data from vac- of base VLPs would be more secure than the classical cine candidates, it would not be realistically feasible to inserting method, which could cause malconformation administer the vaccines to the most needed population if of VLPs and non-exposure of the target antigen by the the manufacturing cost is not financially acceptable. We immune cells of vaccinees. It would be very optimistic must put all our effort into the development by designing to say we would successfully have chimeric HPV/HIV new vaccine models, proposing cheaper production VLP-based vaccine within the next 5 years, but there methods to make vaccines less expensive, more stable, is a great possibility that new VLP production methods and finding optimal HIV immunogens. and other vaccine models for chimeric VLP would be No part of this publication may be reproduced or photocopying without the prior written permission o discovered during the period. Key issues HPV can infect basal epithelial (skin or mucosal) cells and high-risk genotypes, 16, 18, 31, 33, 35, 39, - VLPs have very similar appearance to the original 45, 51, 52, 56, 58, 59, and 66, can cause cervical virus and are normally composed of only virus cancer and other mucosal cancers (e.g., anogenital, structural proteins, which can be displayed in a head and neck). While most HPV infections of the cer- highly repetitive manner. vix are asymptomatic and more than 90% are nor- - Since VLPs do not contain any viral genetic mate- mally cleared within 2 years by host immune system, rial inside, they lack the capacity to replicate and HIV-infected women are more likely to have simultane- are non-virulent, and composing proteins can ous infection with multiple HPV genotypes and are at form VLPs by self-assemble manner and can be high risk for HPV-related cancers than women without enveloped or non-enveloped. HIV55,74. - VLPs are structurally stable and can be manipu- Both HPV and HIV are sexually transmitted patho- lated to display heterologous protein on their sur- gens and delivering chimeric HPV/HIV VLP vaccine face or to carry nanoparticles. mucosally to induce local and systemic immunity - Cervarix® (2009, GlaxoSmithKline) and Garda- against these two pathogens would definitely help to sil®9 (2014, Merck & Co.) are bivalent (genotypes protect humans from prevalent HPV/HIV infections, es- 16 and 18) and 9-valent (genotypes 6, 11, 16, 18, pecially among the young generation. For example, 31, 33, 45, 52, and 58), respectively, and are cur- currently, we need to inject the licensed HPV vaccines rently used to prevent HPV infections. 3 times for a separated period. Moreover, HIV vac- - HPV VLP vaccines are still under investigation to cines, up to the present, also are being designed to cover more genotypes and improve immunoge- introduce a few times over months. The globalization nicity and cross-protection. has become much bigger and people have been mov- - Based on several positive results in preclinical ing around all over the world more often. Considering studies with animal models, HIV VLPs can be safe this fact, vaccinating people with chimeric vaccines and immunogenic in various immunization routes would increase the number of vaccinees by less dos- regardless of coadministration of adjuvants and es, and we will be more protected from those patho- should be continuously researched to acquire an gens through herd immunity. Furthermore, one of the effective HIV vaccine. biggest facts, especially in developing countries, is - The insertion of heterologous protein into the that the people who had the first or second dose often loops located in HPV L1 protein could improve the do not come back for the following dose due to geo- specific immunogenicity of the heterologous pro- graphical, social, and logistic reasons75. In addition, in tein compared to the insertion to other locations Africa, there are more HIV-infected people who are at of L1 protein. higher risk for HPV-related malignancies so that clinical - Critical issues to improve VLP-based vaccine: (i) trials for chimeric HPV/HIV vaccines should be held not formulation level, (ii) administration route, (iii) de- only in North America, Latin America, and Europe but livery vehicles, and (iv) modes of delivery. 230 Eto, et al.: Chimeric VLP-based vaccines for HPV/HIV - A new concept, called VLVs, of merging VLPs and 15. Liu XS, Abdul-Jabbar I, Qi YM, Frazer IH, Zhou J. Mucosal immunisation with papillomavirus virus-like particles elicits systemic and mucosal im- replication-deficient virus vectors can lead both munity in mice. Virology. 1998;252:39-45. 16. Liu WJ, Liu XS, Zhao KN, Leggatt GR, Frazer IH. Papillomavirus virus-like humoral and cell-mediated immunity safely and particles for the delivery of multiple cytotoxic T cell epitopes. 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