Immunization with HIV protease peptides linked to syngeneic erythrocytes
© Boberg et al; licensee BioMed Central Ltd. 2007
Received: 08 December 2006
Accepted: 18 April 2007
Published: 18 April 2007
New potent vaccine adjuvants are desirable for increasing the efficacy of novel vaccine modalities such as DNA and peptides. We therefore tested if syngeneic erythrocytes could serve as delivery vectors for selected HIV peptides and compared the potency of these constructs to immunization with peptides in phosphate buffered saline or in incomplete Freunds adjuvant.
Immunization of mice with peptides in a low dose (5 ng) coupled to erythrocytes induced a weak immune response in mice. These peptides alone (5 μg) gave no immune responses, while formulating the peptides (50 μg) in IFA induced strong homologous immunity as well as prominent cross reactivity to a related mutant epitope. Thus, vaccine delivery using syngeneic erythrocytes, although attractive for clinical use, might be of limited value due to the low amount of antigen that can be loaded per erythrocyte.
Peptide-based vaccines have been shown to be immunogenic in animal models, and well tolerated in man [1, 2]. One major benefit of peptide-based immunogens is the ease with which the vaccine can be modulated in order to fit new variants of a variable microbe such as HIV . One application of such a vaccine strategy would be to target viral mutants conferring escape from antiretroviral drugs. As certain known mutations within epitopes of the HIV proteins are associated with resistance to the drugs  those epitopes variants could be included in a peptide-based vaccine. However these kinds of vaccines may need to be adjuvanted in order to be used in humans. The strategy of using syngeneic erythrocytes as delivery vectors is attractive, since no external compounds are used. Further, red blood cells (RBCs) are naturally removed by macrophages from the bloodstream and thereby targeted to immune cells. In addition, no toxic side effects have been found in host tissue following RBC-antigen delivery . HIV Tat protein coupled to red blood cells was shown to induce as potent immune response as protein formulated in Freund's adjuvant . The RBC method also induced protective immunity in mice and cats lethally challenged with HSV-1 and FIV-M2, respectively [7, 8]. The objective of the present study was to test the efficacy of syngeneic erythrocytes as delivery vectors and adjuvant for peptides deriving from the HIV-1 protease.
Immunization schedule of the study
Site of injection1
Symbol used in Figure 1
4 × 50 μg
Downward pointing triangle
4 × 50 μg
Black filled Diamond
PR75–84 d.mut low2
4 × 5 μg
Blue open Diamond
VLVGPTPVNI biotinylated to synergeneic erythrocytes
2 × 5 ng3 + 2 × 12.5 ng4
VLVGPTPF NV biotinylated to syngeneic erythrocytes
2 × 5 ng3 + 2 × 12.5 ng4
Biotinylated syngeneic erythrocytes
3 × 200 μl + 1 × 500 μl blood
We have previously shown that peptide immunization with the wild type protease epitope, PR75–84 wt, or the mutant variant, PR75–84 d.mut, both formulated in IFA, induces potent immune responses in mice . This suggests that choosing either of these peptides as a component in a vaccine may suppress wild type virus as well as viral variants carrying drug-induced mutations . Based on these findings we compared delivery of the two HIV protease peptides, PR75–84 wt and PR75–84 d.mut, either prepared in emulsion with IFA or coupled to syngeneic erythrocytes. To further increase the uptake of the peptide/erythrocyte complex by antigen presenting cells, the erythrocytes were chemically modified to be recognized as aging by macrophages and thereby cleared more rapidly by macrophages and antigen-presenting cells [5, 10]. A strong immune response was detected after peptide/IFA immunization, whereas only a weak cellular response was measurable upon four consecutive RBC-peptide conjugate immunization. In a parallel experiment, 5 μg of PR75–84 d.mut peptide in PBS was used for immunization and this amount of peptide without adjuvant did not induce any immune responses. These findings suggest that erythrocytes may serve as delivery vectors for very small amounts of peptide. Since the loading capacity of erythrocytes is limited, as well as the volume of blood for each injection, the enhanced presentation of peptides to macrophages by the modified erythrocytes is probably not sufficient to compensate for a small amount of peptide antigen in a clinical scenario.
This work was supported by grants from the AIDS Vaccine Integrated Project (AVIP), European Union and The Swedish Physicians Against AIDS Research Foundation, Sweden.
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