We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75–84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes).
Combinations of antiretroviral drugs are still the only effective approach to delay the progression to acquired immuno-deficiency syndrome (AIDS) in HIV-1 infected patients. Due to the ability of the virus to introduce and tolerate mutations within the viral proteins, resistance to antiretroviral drug treatment may occur [
Interestingly, there seemed to be a good correlation between a theoretical alpha amphipathic region in the C-terminal region and the ability of the constructs to form pentamers.
An epitope from the HIV protease protein corresponding to amino acids 75–84 was selected for genetic conjugation to the B-subunit of the cholera toxin. The reason for this selection is that in an HIV infected individual it may become possible to immunize against development of drug resistance [
Forward and reverse oligonucleotides corresponding to amino acid sequences 75–84, 75–89, and 75–94 from HIV protease harboring the mutations I84V and/or V82F were purchased from SGS DNA, Köping, Sweden. The oligonucleotides were made to encode overhangs corresponding to HindIII and Kpn1 restriction enzyme sites, to enable cloning into an expression vector [
The fusion proteins, hereafter called rCTB-PR75–94I84V
Groups of HLA-A0201 transgenic C57BL/6 mice [
Two weeks following each of the immunizations, individual blood samples were taken. Sera were individually collected and stored at −20°C until use. The peripheral blood mononuclear cells (PBMCs) from group-wise-pooled blood were separated by Ficoll-Paque gradient (GE Healthcare, Uppsala, Sweden). After washing, the PBMCs were resuspended in 1 mL RPMI medium containing 1% PEST, 2 mM L-glutamine, 1% HEPES, and 20% fetal calf serum (referred to as RPMI plus medium).
At the end of the studies, animals were sacrificed by cervical dislocation, and the spleens and serum harvested. Spleens were individually mashed in PBS, and the cell suspensions were separated by Ficoll-Paque gradient and resuspended in 2 mL RMPI plus medium. Cells (PBMCs and splenocytes) were diluted to a final concentration of 2 million cells per mL. One hundred
IFN-
A carboxymethyldextran (CM5; Biacore, Uppsala, Sweden) sensor chip was docked to the BIACORE2000 machine (Biacore, Uppsala, Sweden) and flowed with HBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% Surfactant P20). GM-1 was immobilized on the chip by direct injection, followed by blocking in 1% BSA in HBS-N (10 mM HEPES pH 7.4, 0.15 M NaCl). The samples (rCTB standards, controls, and
Statistical comparisons between groups were performed using the nonparametric Kruskal-Wallis test to detect differences between groups for the ELISpot results. At significant differences, individual groups were compared using the Mann-Whitney
The importance of cytotoxic T cells in HIV infection as well as in SIV/SHIV infection in nonhuman primates is well established [
(a)–(i) Schematic picture of the immunogens.
Under native conditions, we found that rCTB-PR75–89I84V, rCTB-PR75–94I84V, and rCTB-PR75–94V82F/I84V fusion proteins mainly formed pentamers (Figure
Summary of rCTB-proteaseepitope fusion protein immunizations.
Question | Immunized with | Amino acid sequence of linked epitope | Conclusion |
---|---|---|---|
Can the immunogenicity of an epitope be enhanced by linking it to recombinant Cholera toxin |
rCTB-P |
rCTB-VLVGPTPVN |
Linking the HIV derived epitope, P |
rCTB-P |
rCTB-VLVGPTPVN |
||
rCTB + P |
rCTB + VLVGPTP |
||
P |
VLVGPTP |
||
rCTB | rCTB | ||
Untreated | — | ||
| |||
Do we need the pentameric form of the chimeric protein for enhancement of immune response? | rCTB-P |
rCTB-VLVGPTPVN |
Pentamerization, permitting GM-1 binding, of the fusion protein correlated with enhanced immune responses to the attached HIV epitope. |
rCTB-P |
rCTB-VLVGPTPVN |
||
rCTB-P |
rCTB-VLVGPTPVN |
||
rCTB + P |
rCTB + VLVGPTPVN |
||
P |
VLVGPTPVN |
||
Untreated | — | ||
| |||
Can we broaden the immune response by using a variant of the P |
rCTB-P |
rCTB-VLVGPTP |
Addition of the extra mutation reduced the binding property of the fusion protein to GM-1, and resulted in a reduced epitope-specific immune response. |
rCTB + P |
rCTB + VLVGPTP |
||
P |
VLVGPTP |
||
rCTB | rCTB | ||
Untreated | — |
Silver staining of rCTB-protease fusion proteins. Purified protein was run on SDS-PAGE gels and silver stained. Lanes 1–6: native samples. Lanes 7–12: denatured samples. Lanes 1 and 12: see Blue 2 molecular weight standard, Lanes 2 and 7: rCTB standard, Lanes 3 and 8: rCTB-PR75–84I84V, Lanes 4 and 9: rCTB-PR75–89I84V, Lanes 5 and 10: rCTB-PR75–94I84V, and Lanes 6 and 11: rCTB-PR75–94V82F/I84V.
Theoretical prediction of the secondary structure characteristics of the fusion proteins based on the Eisenberg method.
Immunizing with the pentameric rCTB-PR74–94I84V induced significantly higher responses (
Receptor binding property of the CTBa moiety of fusion proteins relative to the binding of unmodified CTB.
Group | Fusion protein | Maximal binding | Actual binding | Percent bindingb (%) |
---|---|---|---|---|
A | rCTB-P |
0.79 mg/mL | 0.79 mg/mL | 100 |
B | rCTB-P |
0.86 mg/mL | 0.64 mg/mL | 74 |
C | rCTB-P |
0.81 mg/mL | 0.75 mg/mL | 93 |
D | rCTB-P |
0.80 mg/mL | 0 mg/mL | 0 |
aCTB: Cholera toxin B subunit.
b(bound fusion protein/maximal bound rCTB protein) * 100%.
Specific immune responses to HIV PR75–84 peptide and variants harbouring drug-resistance mutations. Splenocytes were purified two weeks following the last immunization. The cells were stimulated over night with the (a) singly mutated (I84V), (b) doubly mutated (V82F/I84V), or (c) the wild-type peptide variants of PR75–84. Bars are showing the median response of the different groups. Specific responses were calculated by subtracting background responses to a control peptide from PR-specific immune responses. Proportion of responding animals (>50 spot forming cells/106 splenocytes) are indicated above each individual bar.
Taken together, our results imply that the capacity of CTB fusion proteins to be in a pentameric configuration, which is required for GM-1 binding, is crucial in order to induce cellular immune response against the conjugated epitope. It has been shown that conjugation of antigen to CTB enhances the transport of the antigen over mucosal surfaces and an increased delivery of the antigen to macrophages and dendritic cells [
To investigate the capacity to induce an epitope-specific memory response following immunization with the fusion proteins, we introduced a four-month interruption in the immunization protocol between a third and fourth immunization. The response was measured following three immunizations with rCTB-PR75–94I84V(a response of 223 SFC/106 PBMC, data not shown). During the four-month interruption of the immunization protocol, the level of the response was reduced to 50% of the original level. However, the late fourth immunization at week 28 resulted in a rapid anamnestic response (578 SFC/106 PBMC), indicating that an immunological memory was induced by the initial three immunizations.
Immunization with the rCTB-PR fusion proteins did induce immune responses against not only the epitope variant found in the proteins but also against similar PR75–84 epitope variants (PR75–84 wild type and PR75–84V82F/I84V; Figures
When the CTB molecule was linked to peptides extended by five or ten amino acids downstream, the CTB-peptide complex readily formed pentamers. Even though the added amino acids buried the epitope of interest within a longer (app. 20 amino acids long) stretch of amino acids, it still was possible to elicit an immune response to the epitope of interest. Thus, we show that the key issue of using CTB as a carrier molecule for augmenting the cellular response to an epitope is to retain the natural pentameric structure of CTB. This structure seems to be more affected by the amino acid composition of the epitope of interest than of the length of the epitope attached. It was possible to induce cellular immune responses against the short HIV-1 protease epitope within a longer stretch of amino acids as long as the CTB molecule allowed forming pentamers. This knowledge is important since it provides a tool to evaluate the potential of a fusion protein including CTB as a carrier. Consistently throughout the study, the highest epitope-specific immune response was observed following immunization with rCTB-PR75–94I84V. This fusion protein contained a 20-mer region of the HIV protease, and the CTB moiety retained the ability to form functional pentamers. The fusion protein that contained a 15 amino acid long epitope of HIV protease also induced a good but lower response. These two fusion proteins were found to bind with different affinity to GM-1 and the binding correlated with immunogenicity. Taken together, our results show that peptides from HIV fused to CTB in a manner that allows pentamerization of the protein augments the cellular immune response to the epitope. This concept may be used to induce high and durable cellular immune responses against epitope antigens.
All authors declare that they have no conflict of interests.
The authors would like to thank PhD Susanne Johansson for her assistance in statistical analysis. This work was financially supported by grants from the Europrise program HIV Vaccines and Microbicides, Swedish physicians against AIDS research foundation, the Swedish Research Council, the Swedish Agency for Innovation Systems, and Cancer and Allergy foundation. The founders were not involved in study setup, data interpretation, or finalization of the article.