Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Sep 30;94(20):10856-61.
doi: 10.1073/pnas.94.20.10856.

Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence

J D Ahlers  1 , T Takeshita, C D Pendleton, J A Berzofsky
Affiliations

Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence

J D Ahlers et al. Proc Natl Acad Sci U S A. .

Abstract

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Modification of a T cell helper epitope creates a more potent peptide than the native sequence. (A) Dose–response curves of CD4+ T cell line derived from B10.BR mice (expressing the class II molecule I-Ek) immunized with native T1 peptide and stimulated in vitro with native T1 peptide (○) and modified T1(A)436 (•). The background incorporation in the absence of antigen was 250 cpm. (B) Substituted peptide 436 Ala is immunogenic at lower doses than wild-type T1 peptide for induction of T cell responses to T1. Proliferation normalized to the PPD response of draining lymph node cell cultures from B10.BR mice immunized with T1(solid bars) and T1(A)436 (hatched bars) in CFA and stimulated in vitro with 20 μM native T1 peptide. The range of PPD responses was 32–50,000 cpm, the range of background incorporation was 5500–8000 cpm for the six immunization groups, and the PPD response was always 4- to 7-fold above the background. Comparable results were obtained in three (A) and two (B) independent experiments.
Figure 2
Figure 2
Sequence modification increases immunogenicity for CTL induction in vivo. CTL response of A.AL mice after a single immunization in the adjuvant DOTAP with increasing doses of native vaccine construct PCLUS 3–18IIIB (○) and second generation construct PCLUS 3 (A-436)-I13 (▪) to P18IIIB-pulsed BALB/c 3T3 fibroblast target cells. Mean specific 51Cr release of triplicate cultures ± SEM are plotted vs. effector-to-target cell ratio. Error bars not visible are smaller than the symbols. Lysis of fibroblast target cells without peptide was <15%. The CTL response of animals immunized with the second generation construct PC3(A)I13 was significantly different from that of animals immunized with the native construct P3–18IIIB at an immunization dose of 3 and 10 nmol (P < 0.05, Tukey–Kramer HSD). The second generation construct elicited >17-fold more lytic units at the 3-nmol dose when compared with the native construct at 25% specific lysis. Comparable results were obtained in three independent experiments.
Figure 3
Figure 3
Covalent linkage of the Th and CTL epitopes is required for optimum CTL induction. A.AL mice were immunized with 20 nmol of peptide in the adjuvant DOTAP. Animals were immunized with either the minimum CTL epitope I10, RGPGRAFVTI, a mixture of helper and CTL epitopes T1 plus I10, a mixture of modified helper epitope T1(A) 436 plus I10, covalently linked helper and CTL epitope, PCLUS 3–18IIIB, or PCLUS 3 (A)436-I13. Fourteen days after a single s.c. immunization, bulk spleen cells were stimulated in vitro with 0.5 μM P18IIIB, and 10% Rat T Stim was added 24 h later as a source of interleukin 2. CTL activity was tested 7 days later in a 51Cr release assay at an effector-to-target ratio of 12.5:1 against 0.5 μM P18IIIB-pulsed 18 neo target cells.
Figure 4
Figure 4
Enhancement of T cell help for CTL induction is MHC-linked. CTL response of A.AL (I-Ek, Dd) and A.TH (I-As, Dd) mice to P18IIIB-pulsed target cells. Mice were immunized with 16 nmol of native HIV-1 vaccine construct P3–18IIIB (○) and second generation construct PC3(A)I13 (•) in the adjuvant DOTAP. Spleen cells from two mice of each group were pooled 14 days after a single immunization and stimulated in vitro with 0.5 μM P18IIIB-pulsed spleen cells. Specific lysis of 51Cr-labeled 3T3 fibroblast target cells was measured 7 days after in vitro stimulation. Means of triplicate cultures at each effector-to-target cell ratio ± SEM are shown. Nonspecific lysis of unpulsed targets was <10%. The CTL response of animals immunized with the second generation construct PC3(A)I13 was significantly different from that of animals immunized with the native construct P3–18IIIB in A.AL mice expressing the class II molecule I-Ek (P < 0.05). Comparable results were obtained in three independent experiments.
Figure 5
Figure 5
Epitope enhancement for CTL induction is primarily due to modification of the T helper epitope. CTL response of A.AL mice immunized with 5 and 15 nmol of native construct P3–18IIIB (○), P3-I13 [comprised of the native helper epitope and the truncated CTL epitope I13 (□)], PC3(A)-18IIIB [comprised of the modified T helper epitope and full length CTL determinant P18IIIB (▴)], and PC3(A)-I13 [containing both the modified helper epitope and truncated CTL determinant I13 (⧫)]. Nonspecific lysis of fibroblast targets is indicated by dashed lines. Error bars representing SEM are visible only when greater than the size of the symbol. Animals immunized with constructs containing the modified multideterminant helper peptide PC3(A) had significantly greater specific lysis of P18IIIB-pulsed fibroblast target cells at both immunization doses than animals immunized with vaccine constructs containing the native multideterminant helper peptide (P < 0.05). Comparable results were obtained in two independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Berzofsky J A. Ann NY Acad Sci. 1993;690:256–264. - PubMed
    1. Berzofsky J A. Ann NY Acad Sci. 1995;754:161–168. - PubMed
    1. Pogue R R, Eron J, Frelinger J A, Matsui M. Proc Natl Acad Sci USA. 1995;92:8166–8170. - PMC - PubMed
    1. Bodmer H C, Pemberton R M, Rothbard J B, Askonas B A. Cell. 1988;52:253–258. - PubMed
    1. Lipford G B, Bauer S, Wagner H, Heeg K. Vaccine. 1995;13:313–320. - PubMed