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. Author manuscript; available in PMC: 2022 Nov 29.
Published in final edited form as: Vaccine. 2009 May 3;27(32):4337–4349. doi: 10.1016/j.vaccine.2009.04.032

Human immunodeficiency virus-like particles with consensus envelopes elicited broader cell-mediated peripheral and mucosal immune responses than polyvalent and monovalent Env vaccines

Sean P McBurney 1, Ted M Ross 1,2,*
PMCID: PMC9707700  NIHMSID: NIHMS112041  PMID: 19389453

Abstract

Envelope (Env) sequences from human immunodeficiency virus (HIV) strains can vary by 15-20% within a single clade and as much as 35% between clades. Previous AIDS vaccines based upon a single isolate often could not elicit protect immune responses against heterologous viral challenges. In order to address the vast sequence diversity in Env sequences, consensus sequences were constructed for clade B and clade C envelopes and delivered to the mouse lung mucosa on the surface of virus-like particles (VLP). Consensus sequences decrease the genetic difference between the vaccine strain and any given viral isolate. The elicited immune responses were compared to a mixture of VLPs with Envs from primary viral isolates. This polyvalent vaccine approach contains multiple, diverse Envs to increase the breadth of epitopes recognized by the immune response and thereby increase the potential number of primary isolates recognized. Both consensus and polyvalent clade B Env VLP vaccines elicited cell-mediated immune responses that recognized a broader number of clade B Env peptides than a control monovalent Env VLP vaccine in both the systemic and mucosal immune compartments. All three clade C Env vaccine strategies elicited similar responses to clade C peptides. However, both the consensus B and C Env VLP vaccines were more effective at eliciting cross-reactive cellular immune responses to epitopes in other clades. This is the first study to directly compare the breadth of cell-mediated immune responses elicited by consensus and polyvalent Env vaccines.

Keywords: HIV-1 vaccine, Consensus, Polyvalent, virus-like particles

Introduction

The development of an effective AIDS vaccine has been hampered by the intrinsic diversity among circulating populations of HIV-1 in various geographical locations. There is a need to develop vaccines that can elicit enduring protective immunity to variant HIV-1 strains. Based on viral genetic distances and positions in phylogenetic trees, HIV-1 is divided into three separate groups: M, O, and N [1]. Group O infections are limited to central Africa and Group N infections are found in a small number of patients in Cameroon [2]. The vast majority of viral isolates are designated as Group M and they responsible for the continuing worldwide AIDS pandemic. Due to the diversity in amino acid sequences between isolates within Group M, this group is further subdivided into nine subtypes or clades (A-D, F-H, J and K) [1]. The diversity between isolates in different clades can vary by as much as 35%.

The ultimate goal of an AIDS vaccine is to elicit potent cellular and humoral immune responses that will result in enduring, broadly protective immunity. Over the past 20 years, a number of potential AIDS vaccines have elicited immune responses that decreased viral set-points and maintained CD4+ T cells in vaccinated animals, but sterilizing immunity has not been achieved [3-6]. Often, this limited protection occurs when the challenge virus matches the viral proteins the vaccine [3-6]. However, these artificial challenge models are not reflected in human infections and therefore the diversity of HIV-1 isolates requires vaccine designs that elicit broadly reactive immunity.

The greatest diversity is localized to the viral envelope glycoproteins which may reflect the primary role in eliciting host immune recognition and responses that result in progressive evolution of the envelope proteins during persistent infection. Interestingly, while Env variation is widely assumed to be a major obstacle to AIDS vaccine development, there is very little experimental data in animal or human lentivirus systems addressing this critical issue. One method previously used to address Env variation in an AIDS vaccines is a polyvalent vaccine strategy [4, 7-11]. These vaccines consist of a mixture of divergent isolates of the same antigen administered simultaneously. Polyvalent vaccines function by presenting a wide range of epitopes that cover a majority of individual strains. Vaccines for pneumococcus, poliovirus, and influenza virus have used this strategy [12]. Polyvalent vaccines increase the strength and breadth of humoral immune responses compared to monovalent vaccines [8-10, 13-18]. The increase the number of immunogens, below a certain threshold, does not result in immune interference [19].

More recently, centralized sequences have been developed as an alternative vaccine strategy to address viral sequence diversity. These artificial sequences are designed using computational methods to minimize the distance between a vaccine strain and primary wild-type isolate. The three main strategies for developing centralized vaccines are center of the tree, ancestral, and consensus (for review see [20, 21]). Each of these designs has advantages for vaccine development. Consensus sequences minimize the degree of sequence dissimilarity between vaccine immunogens and circulating virus strains by creating artificial sequences based upon the most common amino acid in each position in an alignment [22-27]. Recently, vaccine strategies utilizing consensus or ancestral HIV-1 Gag and Env sequences have proven potent inducers of CTL activity [23, 28-31]. These consensus Envs form native structures and facilitate infection via the CCR5 coreceptor [32, 33].

In this study, monovalent, polyvalent and consensus Env vaccines were compared for the ability to elicit broadly reactive immune responses following vaccination in mice. Each set of Env vaccines was generated from clade B or clade C envelope sequences. HIV-1 virus-like particles (VLP) were used to deliver these Env sequences to the immune system in their native trimeric structure on the surface of a viral particle and the elicited immune responses were compared for the breadth of Env reactivity.

Materials and Methods

Envelope gene sequences

The wild-type HIV-1 subtype B and C and consensus full-length env gene sequence was derived from the most common amino acids found at each location within the Env gene from over 200 isolates for each clade (Los Alamos National Laboratory; www.lanl.gov). Contemporary subtype B and C env genes were cloned by polymerase chain reaction (PCR) amplification from Envgp160 plasmids obtained from the AIDS Reagent and Reference Program (National Institutes of Health). Primers were designed to insert a 5′ XhoI site and a 3′ NheI site to facilitate cloning into the VLP vector. The nucleotide sequences of the env genes are available under accession numbers PVO.4 (AY835446), AC10.0.29 (AY835446), RHPA4259.7 (AY835447), SC422661.8 (AY835441), Du151.2 (AY835441), CHN19 (AF268277), and ZM214m.PL15 (DQ388516).

DNA Plasmids

The pTR600 vaccine expression plasmid [34] and the HIV-1 VLP expressing plasmid, have been previously described [35]. Briefly, the pHIV-wtVLPADA plasmid encodes for the following gene sequences: HIV-1BH10 gag–pol (pHIVBH10 nt 112–3626) (accession number M1564) and HIV-1ADA vpu, env, rev, tat (nt 5101–8159). Safety mutations were engineered into Gag to prevent viral RNA packaging [36, 37] and RT to prevent reverse transcriptase and RNase H activity (pHIV-VLPADA) [38-40]. Each VLP was expressed from a cytomegalovirus immediate-early promoter (CMV-IE) for initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation signal (BGH poly A) for termination of transcription. Consensus Env VLPs were constructed by substituting ADA env with the consensus env sequences from the consensus clade B and C envelopes (LANL database). The primary isolate VLPs were constructed by substituting ADA env with the primary env sequences. Plasmids expressing the HIV-1NL4-3 Gag gene products only, pGagp55, was derived from codon-optimized sequences (phGag), as previously described [35, 41]. pGagp55 encodes for an immature, unprocessed HIV-1 Gag particle. Each plasmid was amplified in Escherichia coli strain-DH5 alpha, purified using anion-exchange resin columns (Qiagen, Valencia, CA), and stored at −20°C in dH2O. Plasmids were verified by appropriate restriction enzyme digestion and gel electrophoresis. Purity of DNA preparations was determined by optical density reading at a wavelength of 260 and 280 nm.

Purification of virus-like particles

Supernatants from COS cells, transiently transfected with plasmid expressing Gag or VLPs were purified via ultracentrifugation (100,000 × g through 20% glycerol, weight per volume) for 4 h at 4°C. The pellets were subsequently resuspended in PBS and stored at −20°C until use. Protein concentration was determined by Micro BCA™ Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL, USA).

Expression analysis

For Western hybridization analysis, 1 μg of purified VLP was diluted 1:2 in SDS sample buffer (Bio-Rad, Hercules, CA), boiled for 5 min, and loaded onto a 10% poly-acrylamide/SDS gel. The resolved proteins were transferred onto a nitrocellulose membrane (Bio-Rad) and incubated with a 1:500 dilution of HIV-1IIIB rabbit anti-gp120 polyconal sera (Advanced Biotechnologies Inc, Columbia, MD) in PBS containing 0.05% Tween 20 and 5% nonfat dry milk. After extensive washing, bound rabbit antibodies were detected using a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit anti-serum and enhanced chemi-luminescence (Amersham, Buckinghamshire, UK). The western blots were scanned and densitometry was determined using ImageJ software (http://rsbweb.nih.gov/ij/). Expression levels were normalized to ConB VLP as it had the highest expressions levels.

Immunization of mice

Female BALB/c mice (5–7 weeks old) were immunized at weeks 0, 4, and 8 with purified VLPs (20 μg total protein) and co-inoculated with phosphorothioate CpG oligodeoxynucleotides (CpG ODNs, 5 μg each, total of 10 μg) via the nares in a total volume of 30 μL. Each CpG ODN (ODN-1: 5′-TCCATGACGTTCCTGACGTT-3′, ODN-2: 5′-TGACTGTGAACGTTCGAGATGA-3′) [42-46] was synthesized and purified by high-pressure liquid chromatography (Sigma-Genosys, The Woodlands, TX, USA). The CpG ODNs were resuspended in sterile dH2O (2 μg/μl) and stored at -20°C. The composition of each purified VLP preparation is approximately 80-90% Gag gene products and therefore, mice were administered ~0.5-1.0 μg of Env per VLP immunization. Mice were housed in compliance with U.S.D.A. regulations and were monitored daily for weight loss, behavior, and adverse reaction. Mice were partially anesthetized with xylazine (20 mg/ml) and ketamine (100 mg/ml) administered subcutaneously in the abdomen prior to immunization.

Collection of samples

Blood samples were collected by retro-orbital plexus puncture on weeks 0, 2, 6, and 10 post-immunization on anesthetized mice. Sera samples were collected by centrifugation (5,000 rpm, 10 min) and stored at -20°C. Spleens were harvested from vaccinated mice at week 10, and splenocytes were isolated for ELISpot assays, as previously described [47, 48]. Briefly, splenocytes were depleted of erythrocytes by treatment with ammonium chloride (0.1 M, pH 7.4). Following thorough washing with PBS, cells were resuspended in RPMI medium. Cell viability was determined by trypan blue exclusion staining.

Antibody response to VLP immunizations

Serum samples and lung washes were individually collected and tested for antibody (IgG) responses to homologous VLP by ELISA. Each well of a 96-well plate was coated with 50 ng per well of the respective VLP (4°C for 16 hr). Plates were blocked (25°C for 2 hr) with PBS containing Tween 20 (0.05%) and nonfat dry milk (5%) and then incubated with serial dilutions of each sample (25°C for 2 hr). Following thorough washing in PBS-Tween 20 (0.05%), samples were incubated (25°C for 1 hr) with biotinylated goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) diluted 1:5000 in PBS-Tween 20 (0.05%) and nonfat dry milk (5%). The unbound antibody was removed, and the wells were washed. Samples were incubated with TMB substrate (1 hr), and the colorimetric change was measured as the optical density (O.D., 405nm) by a spectrophotometer (BioTek Instruments, Winooski, VT, USA). The O.D. value of naïve sera was subtracted from the samples using antisera from vaccinated mice. Results were recorded as the arithmetic mean ± the standard deviation.

Neutralization assay

Antisera from vaccinated mice were tested for the ability to neutralize virus infection in vitro using TZM-Bl cells indicator cells [49, 50]. These cells express human CD4 (hCD4), human CCR5 (hCCR5), human CXCR4 (hCXCR4), and a luciferase reporter driven by the HIV-1 LTR TZM-Bl cells were cultured in cDMEM with 10% fetal calf serum (10%) (Atlanta Biologicals, Atlanta, GA, USA). Infectivity was determined using serial dilutions of antisera with cells in complete, non-selective media in the presence of DEAE dextran (20 μg/ml) (25°C for 1hr). Cell lysates were harvested in lysis buffer (25mM Tris phosphate, pH=7.8, 2mM DTT, 2mM 1-2-diaminocyclohexane-N, N, N′, N′-tetraacetic acid, 10% gylcerol, 1% Triton X-100) (48 h) and then clarified by centrifugation. Virus neutralization by mouse antisera was determined by measuring the relative light units (RLU) using a Femtomaster FB12 Luminometer (Zylux, Maryville, TN). Neutralization by naïve sera and sera from mice vaccinated with virus-like particles composed of only Gag gene products (no Env) was subtracted from the RLU from assays using antisera from vaccinated mice.

ELISpot assays

The number of anti-Gag and anti-Env specific murine INF-γ (mINF-γ) secreting splenocytes and lung cells was determined by enzyme-linked immunospot (ELISpot) assay (R & D Systems, Minneapolis, MN, USA). Briefly, precoated anti-mIFN-γ plates were incubated (25°C for 1 h) with cRPMI (200 μl) and then were incubated with splenocytes (5 × 105/well) or lung cells (1 × 106/well) isolated from vaccinated mice. Cells were stimulated (48 h) with peptides (15mers overlapping by 11 amino acids) representing the consensus clade B HIV-1 Gag, consensus clade B, or consensus clade C Env proteins (NIH ARRRP). IL-2 was added to all wells (10 units/ml). Control wells were stimulated with PMA(+) (50 ng)/ionomycin (500 ng) or were mock stimulated(-). Plates were washed with PBS-Tween (3×) and were incubated (37°C for 48 h; 5% CO2) with biotinylated anti-mIFN-γ and incubated (4°C for 16 h). The plates were washed and incubated (25°C for 2 h) with strepavidin conjugated to alkaline phosphatase. Following extensive washing, cytokine/antibody complexes were incubated (25°C for 1 h) with stable BCIP/NBT chromagen. The plates were rinsed with dH2O and air dried (25°C for 2 h). Spots were counted by an ImmunoSpot ELISpot reader (Cellular Technology Ltd., Cleveland, OH, USA).

CD4 cell depletion

CD4 cells were depleted from single cell suspensions of splenocytes from individual mice. Depletions were completed using the CD4 (L3T4) microbeads and an autoMACS Separator per manufacturer's instructions (Miltenyi Biotec Inc., Auburn, CA).

Results

Selection of wild-type and design of consensus B or C env gene sequences

The aim of this study was to compare the breadth of immune responses elicited by wild-type (individually or in a mixture) or consensus envelope sequences. The consensus (Con) B or C Env sequences were generated by selecting the most common amino acid at each position of a full-length Env alignment derived from 200 subtype B or C viruses deposited in the 2005 HIV Sequence Database. Each set of subtype sequences were supplemented with env sequences used in a standardized neutralizing panel [51, 52].

The wild-type sequences used to design monovalent or polyvalent vaccines were selected based upon 4 criteria: route of transmission, weeks after transmission, place of transmission, and neutralization phenotypes. All Envs were chosen from isolates from patients that received the virus via sexual transmission. Five of the 7 isolates were transmitted by heterosexual transmission (M-to-F or F-to-M) and 2 isolates by M-to-M transmission (Table 1). Two of the clade B viruses were isolated from patients living in United States, one in the Caribbean, and one in Europe, whereas the three clade C isolates were collected from patients living in Africa and Asia. All of the isolates used in the polyvalent vaccines were collected in the acute phase of infection and were neutralization resistant [51, 52]. Fig. 1A and B depicts an alignment of Con B and Con C Envgp160 sequences and the deduced protein sequences of the contemporary wild-type env sequences. One of the strains from each polyvalent vaccine was used as a monovalent vaccine control. Primary and consensus clade B and C Envs efficiently bound to human CD4, was CCR5-tropic and facilitated infection with similar efficiency (data not shown).

Table 1. Characteristics of Envelopes used in the Virus-like Particle Vaccines.

Vaccine Isolate Name Geographic Location of Patient Mode of Transmissiona Length of Infectionb
Gagp24 --- --- --- ---
Consensus B (Con B) --- --- --- ---
Polyvalent B (Poly B) PVO.4 Italy M-M 4 weeks
RHPA4259.7 USA M-F < 8 weeks
SC422661.8 Trinidad F-M 4 weeks
AC10.0.29 USA M-M 4 weeks
Monovalent B PVO.4 Italy M-M 4 weeks
Consensus C (Con C) --- --- --- ---
Polyvalent C (Poly C) DU151 South Africa M-F 6 weeks
ZM214M Zambia F-M <13 weeks
CHN19 China --- ---
Monovalent C DU151 South Africa M-F 6 weeks
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a

direction of sexual transmission: M-M male to male, M-F male to female, F-M female to male

b

length of infection is defined as time between last negative HIV-1 test and first positive HIV-1 test.

Figure 1. Generation of consensus (Con B and Con C) envelopes.

Figure 1

Figure 1

Figure 1

Figure 1

Figure 1

Figure 1

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Alignment of the deduced protein sequence of full-length (unmodified) Con B Env with those of recently transmitted, contemporary subtype B and C Env isolates. Sequences are compared to consensus gp160, with dots indicating sequence identity, and dashes indicating gaps introduced for optimal alignment. Potential N-linked glycosylation sites are in italics, bolded and underlined. The locations of signal peptide (SP) and gp120/gp41 cleavage sites are indicated, as are the positions of the variable loops (V1–V5) and the transmembrane (TM) domain. (A) Con B. (B) Con C.

Env sequences were cloned into the HIV-1 VLP expressing plasmid that contained the HIV-1BH10 gag–pol and HIV-1ADA vpu, env, rev, tat [35]. Following purification, each particle contained similar amounts of envelope (Fig. 2). The expression of Envgp160 from each VLP vaccine was determined to ensure that similar doses of Env were on the particle surface and delivered with each VLP.

Figure 2. Envelope Expression on Virus-like Particles.

Figure 2

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(A) Purified virus-like particles were loaded with 1 μg of total protein in each well in a SDS-polyacrylamide gel under reducing conditions and probed with rabbit anti-HIVIIIB gp120 polyclonal sera. 1. ConB, 2. AC10.0.29, 3. PVO.4, 4. RHPA4259.7, 5. SC422661.8, 6. ConC, 7. CHN19, 8. Du151, 9. ZM214M (B) The level of envelope expressed in the VLP was determined by comparison of the density of the bands observed in the western blot. Levels are shown as percent of envelope as compared to ConB.

Antibodies elicited by VLP vaccines

Groups of mice (BALB/c) were vaccinated with either 1) the consensus Env VLPs (Con B or Con C) individually, 2) wild-type Env VLPs individually (monovalent: PVO.4 or DU151), or 3) a mixture of wild-type Env VLPs (Poly B or Poly C). As a control, viral particles composed of Gagp24 only (no Env) were administered. All vaccines elicited high-titer serum antibodies that recognized the VLP (1:50,000-1:100,000) at week 10 post-vaccination (Fig. 3A). In addition, anti-VLP IgG antibodies were detected in the lung wash from all mice (Fig. 3B). Neutralizing antibodies were detected in the serum from all vaccinated groups (1:40-1:80). However, these titers were the same as those detected in mice vaccinated with Gagp24 particles.

Figure 3. Antibody Responses.

Figure 3

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(A) Anti-VLP titers were determined by ELISA with 96-well plates coated with matched VLPs at 50 ng per well. Week 10 sera were tested starting at a dilution of 1:200. Endpoint was assigned as the dilution at which background was reached. (B) Week 10 lung washes were used to determine anti-VLP titers in the mucosa. Washes were tested starting at a dilution of 1:10.

Elicitation of Cellular Immune Responses

Mice vaccinated with either consensus Env VLPs and wild-type Env VLPs (monovalent or polyvalent) had splenocytes that secreted INF-γ following stimulation with Env-specific peptides (Fig. 4A and B). There were no Env-specific immune responses elicited in mice that were vaccinated with the Gagp24 particles. Pools of peptides representing each region of Envgp160 were matched to the subtype (i.e. subtype B peptides were used to stimulate subtype B vaccines). Mice had anti-Env splenocyte responses throughout all regions of Env in both clade B (300-800 spots per 106 splenocytes) and clade C (100-400 spots per 106 splenocytes). There was no statistical difference in the number of spots elicited between consensus Env VLPs and polyvalent or monovalent Env VLP vaccines in either subtype. Interestingly, there were differences in the cellular responses elicited in the lung mucosa (site of vaccination) compared to the responses detected in the spleen. All three vaccine strategies (consensus, polyvalent, and monovalent) elicited a high number of responses to the V3 region for both subtypes (Fig. 4C and D). However, mice vaccinated with Con B VLPs had additional responses to C1, C3, V4-C5, ectodomain of gp41 (gp41ecto), the transmembrane (TM) and amino terminal region of the intracytoplasmic domain (gp41TM/ICD N′). In contrast, mice vaccinated with Poly B VLPs had significant responses to only gp41TM/ICD N′ and carboxyl region of the intracytoplasmic domain (gp41ICD C′). The monovalent PVO.4 VLP elicited mucosal responses against V4-C5, gp41TM/ICD N′ and gp41ICD C′ regions. Similar responses were observed using the subtype C VLP vaccines (Fig. 4D).

Figure 4. Anti-Envelope Cell-mediated Immune Responses.

Figure 4

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T-cell immune responses induced by Consensus, Polyvalent and Monovalent clade B and C vaccines. Splenocytes (A&B) and Lung cells (C&D) were isolated and stimulated with overlapping clade-matched envelope peptides. Peptides were separated into approximately equal sized pools of peptides representing the different region of envelope. Responses are represented as SFU per million cells. The values of each column are the mean ± standard deviation.

Envelope Cellular Epitopes

Individual peptides responsible for eliciting cellular immune responses in each peptide set were identified using matrix format peptide pools and then confirmed by testing the peptides individually [53]. Thirty-five individual peptides were identified in mice vaccinated with the Con B VLP against the subtype B peptide set and 35 cross-reactive peptides were identified against the subtype C peptide set (Fig. 5 and Table 2). Similar number of peptides was detected in mice vaccinated with the Poly B VLPs, however, almost no peptides were identified using the monovalent PVO.4 VLP. Interestingly, at least 15 individual peptides were identified in mice vaccinated with the subtype C VLP vaccines against both subtype B and subtype C peptides sets. Mice vaccinated with the Poly C had similar number of identified peptides (15 against subtype B and 17 against subtype C). The monovalent DU151 VLP vaccinated mice had comparable number of identified peptides as Poly C against subtype B peptides (20), but an increase number of identified subtype C peptides (32). Interestingly, mice vaccinated with the Con C VLP had twice as many individual peptides identified (50) against the subtype B peptide set as the subtype C peptide set (23).

Figure 5. Identification of Envelope-specific Peptides.

Figure 5

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Individual envelope peptides were identified by ELISpot using peptide pools in a matrix format. Epitopes were considered positive if they were at least two fold higher than responses observed with Gag only vaccinations. Total number of epitopes recognized by each vaccine in both ConB and ConC envelope peptide sets.

Table 2.

Env peptides individually identified by VLP vaccines.

Con Ba Total
Con Bb 8766 8774 8779 8787 8791 9799 8806 8807 8814 8818 8826 8834 8839 8847 8851 8859 8866 8867 35
8874 8878 8886 8894 8897 8905 8909 8917 8925 8928 8936 8940 8948 8956 8957 8965 8969
Con Cb 9186 9187 9189 9190 9191 9193 9199 9201 9202 9203 9205 9215 9217 9219 9221 9231 9233 9249 35
9253 9261 9265 9277 9278 9281 9282 9290 9293 9294 9306 9310 9322 9369 9373 9381 9385
Poly Ba
Con Bb 8764 8769 8780 8782 8784 8793 8813 8823 8831 8855 8869 8875 8883 8891 8912 8915 8920 8929 28
8931 8935 8941 8943 8944 8949 8951 8964 8972 8973
Con Cb 9185 9193 9197 9200 9205 9219 9223 9233 9245 9253 9257 9265 9274 9282 9286 9293 9294 9308 32
9312 9322 9334 9341 9342 9345 9346 9354 9368 9370 9372 9374 9382 9395
PVO.4a
Con Bb 8971 1
Con Cb 9364 9368 2
Con Ca
Con Bb 8763 8766 8768 8777 8779 8782 8784 8789 8791 8797 9799 8813 8815 8821 8823 8837 8839 8849 50
8850 8851 8852 8857 8859 8866 8868 8873 8875 8878 8880 8883 8886 8888 8899 8902 8904 8911
8912 8919 8928 8935 8940 8941 8943 8948 8957 8959 8964 8969 8971 8972 23
Con Cb 9188 9190 9202 9226 9250 9255 9257 9262 9275 9277 9279 9281 9284 9286 9291 9293 9304 9308
9310 9320 9322 9377 9395
Poly Ca
Con Bb 8777 8789 8791 8817 8837 8839 8849 8877 8879 8911 8926 8928 8966 8968 8969 15
Con Cb 9199 9208 9210 9239 9245 9261 9274 9290 9301 9305 9330 9332 9363 9365 9379 9381 9394 17
DU151a
Con Bb 8763 8791 8807 8819 8821 8833 8839 8849 8853 8855 8862 8865 8877 8882 8883 8896 8911 8913 20
8927 8939
Con Cb 9191 9207 9214 9222 9231 9238 9239 9243 9245 9251 9253 9262 9267 9269 9270 9291 9293 9299 32
9301 9303 9311 9327 9332 9334 9340 9351 9356 9359 9380 9388 9392 9394
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a

vaccine group

b

Env peptide set, numbers represent the NIH AIDS Research and Reference Reagent Program catalog number for each identified peptide

The identified epitopes were mapped to each region of Envgp160 (Fig. 6). Since both subtype B and subtype C peptide sets use overlapping peptides, it was important to determine the percent of the Env covered by these responses and not just the number of peptides identified. The Con B and Poly B VLP vaccines clearly demonstrated increased coverage of both subtype B and C peptide sets compared to the monovalent PVO.4 VLP (Fig. 6 and Table 3). Mice vaccinated with the Con B or Poly B VLP vaccines elicited cellular responses that covered 40-50% of the envelope with equal distribution throughout the protein. Mice vaccinated with Con C VLPs elicited similar coverage as the Con B VLP (~61%) as detected by the subtype B peptide set (Fig. 6), however, only half as much of Env was covered when the subtype C peptide set used for analysis, with almost no recognition of the peptides in the Envgp41. Both the Poly C and DU151 VLP vaccines elicited responses that recognized less of the envelope than the Con C vaccine when tested using the subtype B peptide set. However, the monovalent DU151 VLP had more coverage of the Env than Con C or Poly C VLPs when splenocytes were stimulated using the subtype C peptide set.

Figure 6. Env Regions Covered by Elicited Env Cell-mediated Immune Responses.

Figure 6

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Indentified epitopes were mapped to the appropriate location within Env. Epitopes are shown as boxes of the length covered. When overlapping peptides were identified the entire area covered by both peptides was shown. The total percent of envelope covered is shown as a percent on the left. Total area was calculated by dividing the total number of amino acids within identified epitopes by the total length of Env.

Table 3. Number of Peptides Identified per region of Env.

Con Ba C1 V1V2 C2 V3 C3 V4 C4 V5 C5 Ecto Tm Total
Con Bb 5 3 4 1 3 0 2 2 1 5 9 35
Con Cb 11 5 3 1 4 1 3 0 2 1 4 35
Total 16 8 7 2 7 1 5 2 3 6 13 70
Poly B a C1 V1V2 C2 V3 C3 V4 C4 V5 C5 Ecto Tm Total
Con Bb 5 1 3 0 1 0 1 1 1 3 12 28
Con Cb 5 2 4 0 2 2 2 0 1 3 11 32
Total 10 3 7 0 3 2 3 1 2 6 23 60
PVO.4 a C1 V1V2 C2 V3 C3 V4 C4 V5 C5 Ecto Tm Total
Con Bb 0 0 0 0 0 0 0 0 0 0 1 1
Con Cb 0 0 0 0 0 0 0 0 0 0 2 2
Total 0 0 0 0 0 0 0 0 0 0 3 3
Con C a C1 V1V2 C2 V3 C3 V4 C4 V5 C5 Ecto Tm Total
Con Bb 9 2 4 2 6 0 2 4 3 5 13 50
Con Cb 3 1 3 1 4 2 2 0 3 2 2 23
Total 12 3 7 3 10 2 4 4 6 7 15 73
Poly C a C1 V1V2 C2 V3 C3 V4 C4 V5 C5 Ecto Tm Total
Con Bb 3 0 1 2 1 0 0 2 0 1 5 15
Con Cb 3 0 2 1 1 0 1 1 1 2 5 17
total 6 0 3 3 2 0 1 3 1 3 10 22
DU151 a C1 V1V2 C2 V3 C3 V4 C4 V5 C5 Ecto Tm Total
Con Bb 2 1 3 1 3 1 1 1 2 3 2 20
Con Cb 2 3 6 1 3 0 2 3 1 3 8 32
Total 4 4 9 2 6 1 3 4 3 6 10 52
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a

vaccine group

b

Env peptide set, numbers represent the total number of peptides identified within each given region of Env

To determine the subpopulation of T cells elicited by these vaccines, splenocytes were depleted of CD4+ T lymphocytes. Less than 10% of the remaining splenocytes were CD4 positive. The most reactive common epitopes from both peptide sets were tested against the CD4+ depleted splenocytes. This subset of peptides elicited similar numbers of IFN-γ producing spots to each of the epitopes tested (Fig. 7). No spots were detected over background indicating that the cell mediated immune responses elicited by these vaccines were CD4-restricted.

Figure 7. Identified Epitopes Require CD4+ T Lymphocytes.

Figure 7

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The most common and reactive epitopes from ConB and ConC envelope were used to determine if the identified epitopes were CD4 or CD8. Splenocytes were isolated and stimulated with individual peptides (8 ConB and 7 ConC) either with or without CD4 cell depletion. Responses are represented as SFU per million cells. The values of each column are the mean ± standard deviation.

Discussion

The intrinsic diversity among circulating populations of HIV-1 in various geographical locations is one of the greatest challenges for developing an effective AIDS vaccine. There is a great need to develop vaccines that can elicit enduring protective immunity to variant HIV-1 strains. While variation is observed in all of the viral proteins, the greatest diversity is localized to the viral envelope glycoproteins, evidently reflecting the predominant role of these proteins in eliciting host immune recognition. In this study, we examined the effectiveness of two vaccine strategies (polyvalent and consensus) for eliciting broadly reactive immune responses against Envs from diverse clade B and C isolates. Virus-like particles were used to deliver these envelopes in a native, trimeric structure [54] to a mucosal surface that effectively elicited cellular immune responses. Each VLP vaccine expressed similar amounts of envelope on the particle surface (Fig. 2) and therefore differences in the elicited immune responses were due to the particular envelope and not the amount of Env antigen delivered.

All VLP vaccines were highly immunogenic with each VLP vaccine eliciting neutralizing antibodies against a panel of viral isolates. However, viral Gagp24 particles elicited similar neutralizing titers indicating that a majority of the neutralization activity was most likely directed to cellular proteins embedded in the viral membrane and were not Env-specific. Similar results were also observed following vaccination with VLPs or inactivated virus in rabbits and guinea pigs (personal observations and [55]). The VLPs used in this study were produced in non-primate cells and therefore the particle membrane was coated with host cellular proteins. These proteins would be foreign to the mouse immune system and thereby elicit strong immune responses. While this may be arguably beneficial for a VLP vaccine, it limited the ability to determine the effectiveness of a polyvalent or consensus Env vaccine strategy to elicit broadly reactive neutralizing antibodies. Therefore, future studies in primates, possibly using DNA or viral vectors expressing the Envs from a VLP in vivo, will be necessary to answer these questions.

Each VLP vaccine elicited strong anti-Env cell-mediated immune responses as detected by both the Con B and Con C Env peptide sets. These sets were chosen since they represented all of clade B and C viruses instead of any single isolate. The Con B sequence used in the peptide set was ~90% identical to the four Envs sequences used in the Poly B Env VLP vaccine (Table 4) and they were ~82% identical to the Con C peptide set. Similar results were observed with the clade C isolates to the Con C peptide set. Both consensus and polyvalent Env vaccine strategies elicited cell-mediated immune responses throughout all regions of Env. However, the number of specific epitopes elicited by Con B and Poly B Env VLPs was significantly higher than the monovalent clade B Env VLP (Fig. 5). No matter how potent a response to a particular epitope in envelope, it is not advantageous if the vaccine responses are focused on epitopes that are retained only in a small percentage of HIV-1 isolates [22, 27]. Focusing vaccine responses on epitopes that have escaped and are rare in the current population of virus isolates may be a disadvantage for an effective vaccine. Con B and Poly B Env VLPs elicited responses that recognized 70 and 60 different peptides and covered 48.5 % and 44% of the envelope, respectfully. The monovalent clade B Env VLP recognized only 3 peptides and covered 2.55% of the envelope epitopes. In contrast, monovalent clade C Env VLP elicited cell-mediated immune responses as efficiently as Con C or Poly C Env VLPs against the Con C peptides. Interestingly, the Con B and Poly B Env VLP vaccines had greater or equal coverage of the Con C envelope than any of the clade C vaccines tested (Fig. 6). This is likely due to the higher reactivity observed with the clade B vaccines than the clade C vaccines. However, the Con C Env VLP elicited more cross-reactive responses that covered more clade B epitopes than either Poly C or monovalent clade C Env VLP vaccines.

Table 4. Percent Sequence Homology with Env Peptide Sets.

Env sequences/ Env Peptides Con B Env Peptides Con C Env Peptides
Consensus B 99.0 84.5
PVO.4 90.0 81.4
AC10.0.28 89.6 81.2
RHPA4259.7 91.1 82.9
SC422661.8 91.4 82.2
Consensus C 84.0 97.5
DU151 82.5 93.0
CHN19 81.5 88.4
ZM214 85.3 89.3
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We were concerned that using consensus peptide sets to compare consensus to polyvalent Env vaccine strategies would bias our results towards a consensus Env vaccine strategy. These peptide sets were chosen because they were readily available from the NIH AIDS Reference and Reagent Program. Interestingly, the Con B peptide set was not more likely to detect specific epitopes elicited by a Con B Env antigen compared to a mixture of Envs. In addition, similar results were observed using a peptide set derived from the clade B isolate, SF162 (data not shown). This was not surprising, since the SF162 had ~94% identical amino acids as the Con B sequence.

These VLP vaccines were administered intranasally to elicit mucosal immune responses. Intranasal immunization of VLPs induced both systemic and mucosal immunity. Mucosal infection serves as the major route of HIV infections in the world [56]. While circulating virus and virus-specific immune responses are detected in the periphery shortly after infection, virus-specific immune responses at mucosal sites are critical for the control of infection in many individuals exposed to HIV-1 [57-59]. One advantage of using VLPs, compared to the low or undetectable level of immune responses elicited by soluble antigens delivered intranasally [60], is the administration of these VLPs, with CpG oligodeoxynuceotides as an adjuvant, enhances both systemic and mucosal immune responses [61-66]. This is most likely due to phagocytosis of the VLP immunogen by microfold epithelial cells (M cells) in the nasal lumen that leads to direct deposition of antigen to the nasal associated lymphoid tissue (NALT) via M cell transcytosis [67]. This process results in the induction of strong local (NALT), as well as distant immune responses in both peripheral and mucosal immune compartments [68].

Interestingly, there were differences in the regions of Env recognized by mucosal T cells using these vaccines compared to peripheral T cells (Fig. 4). All the vaccines elicited responses against V3 and gp41 regions of Env. However, the Con B and Con C Env VLPs elicited a broader recognition of Env regions in the mucosa than the monovalent or polyvalent Env VLP vaccines. The elicitation of a broader mucosal cell-mediated immune response by the consensus Envs may allow for recognition of a larger number of isolates at the site of infection. Future studies in non-human primates will needed to determine if these VLP vaccines, administered intranasally, elicit vaginal mucosal immune responses. Intranasal immunization directly stimulates the vaginal mucosal immune system to elicit high titer responses [69-71] and therefore, VLP administration may be an effective strategy to elicit genital mucosal responses.

The aim of this study was to evaluate the breadth of coverage generated by consensus and polyvalent envelopes in virus-like particle vaccines. The breadth of coverage was evaluated using consensus B and C envelope peptide sets. All vaccines were able to generate equal levels of antibody and cellular responses systemically, as well as mucosally. These qualtity of these responses were not equal. Both the Con B and Con C VLP vaccine generated responses that recognized a greater number of Env epitopes thereby creating a greater coverage of Env than either polyvalent vaccine. These results indicate that a consensus Env VLP vaccine would likely provide the greatest protection against a wide range of isolates as compared to a polyvalent vaccine.

Footnotes

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