Oral Abstract Session 01 - Global HIV Vaccine Enterprise

POSTERS
Posters
Topic 1: Adjuvants, Immunogens and Inserts
P01.19 LB
CCL28 Induces Mucosal Homing of HIV-1-Specific Iga-
Secreting Plasma Cells in Mice Immunized With HIV-1
Virus-Like Particles
V. Rainone 1 , G. Dubois 2 , V. Temchura 3 , K. Uberla 3 , M. Nebuloni 1 ,
E. Lauri 1 , D. Trabattoni 1 , F. Veas 2 , M. Clerici 4
1 University of Milan, Milan, Italy; 2 Faculty of Pharmacy,
University of Montpellier, Montpellier, France; 3 Ruhr
University Bochum, Bochum, Germany; 4 Don G. Gnocchi
Foundation, Milan, Italy
Background: MEC/CCL28 (CCL28) binds to CCR3 and CCR10 and
recruits IgA-secreting plasma cells (IgA-ASCs) in the mucosal
lamina propria. Virus-like Particles (VLPs) are a novel vaccine
approach based on non-pathogenic particles that mimic the
structure of virus particles with effective induction of both arms
of the immune response. The suitability of CCL28 as an adjuvant
for the elicitation of optimal mucosal and systemic immunity was
assessed in mice immunized with HIV-1 VLPs.
Methods: Balb/c mice were immunized intramuscularly with a
prime-boost regime based on VLP containing gp160 from HIV-
1 IIIB in the presence/absence of CCL28 and of the parental
control CCL19. Flow citometry evaluation of CCR3 and CCR10
expression was performed on purified splenocytes. Th1 and Th2
cytokine production was performed on splenocytes and either
colon, lungs or uterine cervix, whereas antigen-specific IgG and
IgA antibodies were evaluated in sera and mucosal secretions
by ELISA. Immune sera and mucosal secretions were tested for
ex vivo neutralization activity against HIV-1 either subtype B or
C strains. IgA-ASC recruitment at the mucosal level was verified
with immune-histochemistry.
Results: The following parameters were significantly augmented
in VLP-CCL28 mice compared to control groups: the percentage
and the surface density of CCR3 and CCR10 on CD19+
splenocytes; IFN-γ, IL-4 and IL-5 production in splenocytes
and mucosal specimens; total IgA titers in sera and in mucosal
secretions; antigen-specific IgG and IgA titers in sera and in
mucosal secretions. Sera and mucosal secretions from VLP-CCL28
mice showed a significantly augmented neutralizing activity
against homologous and heterologous viruses. IgA-ASCs were
significantly increased in mucosal tissues of VLP-CCL28 mice.
Conclusion: CCL28 used as an adjuvant has a robust
immunomodulatory effect on potentially beneficial mucosal and
systemic immune responses. These findings suggest that CCL28
could play a useful role in increasing the efficacy of preventive
vaccines for mucosally transmitted viral infections.
104
AIDS Vaccine 2012
P01.20 LB
NMR Spectroscopy of HIV-1 gp120 Outer Domain
M. Sastry 1 , L. Xu 1 , S. Bhattacharya 2 , G.J. Nabel 1 , C.A. Bewley 3 ,
P.D. Kwong 1
1 Vaccine Research Center, NIAID/NIH, Bethesda, MD, USA;
2 New York Structural Biology Center, New York, NY, USA;
3 NIDDK, National Institutes of Health, Bethesda, MD, USA
Background: The outer domain (OD) of HIV-1 gp120 has been
proposed as a minimal immunogen to elicit broadly neutralizing
antibodies. However, OD is heavily glycosylated, contains many
flexible regions, and immunization with a number of different OD
variants has thus far failed to elicit neutralizing antibodies. An
understanding of the conformational space sampled by the OD
in its unliganded state, however, may assist in the use of OD as
an immunogen.
Methods: We developed a method to isotopically enrich
glycoproteins using a mammalian expression system that exploits
the high level of protein expression obtained from an adenoviral
vector, and employed heteronuclear NMR spectroscopy to
obtain structural and dynamic information of unliganded OD.
Multidimensional NMR experiments were recorded on uniformly
labeled 15N/ 13C OD as well as on samples selectively enriched
in 15N-labeled Gly, Ile, Leu and Val. Experiments for backbone
assignments were also recorded on an OD sample enriched in
15N/13C for Ile, Leu and Val.
Results: We successfully produced isotopically labeled OD
samples, suitable for NMR analysis. We also identified Gly, Ser,
Val, Leu and Ile residues using samples selectively enriched in
15N for Gly, Val, Leu and Ile. Standard triple resonance NMR
experiments on the isotopically labeled OD were combined with
backbone experiments recorded on a second sample – that was
selectively enriched in 15N/ 13C for Ile, Leu and Val – to assign
HN, C’, C and N backbone resonances in about 80 of the 220
alpha
residues of OD.
Conclusion: We succeeded in assigning ~1/3 of the backbone for
unliganded OD with triple resonance experiments. Extension of
these assignments with NOESY experiments is now proceeding.
Our results indicate that a solution structure of the highly
glycosylated HIV-1 gp120 OD is feasible.