FIV research

1. Presentation on FIV Review articles
Feline Immunodeficiency Virus (FIV) as A Model for Study of
Lentivirus Infections and Vaccination: Parallels with HIV
Presented By:
Kutub Ashraf ,M1 Student
PAVAL Lab ,UJF, France 25/9/2015

2. FIV in Short………
• FIV is a significant pathogen in the cat in addition, it is the smallest
available natural model for the study of lentivirus infections.
• Although divergent at the amino acid level, the cat lentivirus has an
abundance of structural and pathophysiological commonalities with HIV .
• Due to marked similarity in genomic organization, virus structure, virus
replication and disease pathogenesis of FIV and HIV, infection of cats with
FIV is a useful tool to study and develop novel drugs and vaccines for HIV.
Interstingly, FIV has about 51% nucleic-acid(nc) identity and 29% amino-acid
(aa) identity to HIV-1 at the viral structural and enzymatic proteins

3. Pathogenesis of FIV
• The virus infects lymphocytes in which it establishes a persistent infection.
An immune response develops that includes both cell-mediated and
humoral immunity, which keeps the virus under control for long periods of
time.
• Once infected, cats do not recover from FIV infection because the virus lies
dormant within certain cells where it is hidden from the immune response.
Many FIV-infected cats live healthy lives but some develop
immunodeficiency and eventually die of a variety of clinical syndromes.
• However, in contrast to the primate lentiviruses, FIV does not use CD4 as a
primary receptor and displays broader cellular tropism in vivo, infecting a
large number of B and CD8+ T cells, as well as CD4+ T cells and
macrophages

5. The main problems of
vaccination
A vaccine has to be very powerful in order to prevent the
infecting FIV from invading cells and establishing a persistent
infection
FIV occurs in 5 subtypes. If these subtypes are also different
antigenic types, a vaccine would have to protect against the
predominant subtype in a particular geographic area.

6. • Does the existing vaccine protect cats from field virus?
Simply no, Although the vaccine has been shown to protect against
laboratory strains of virus initially but it is not clear if it protects against
FIV strains that are present in the field, which may be of higher
virulence.
• The vaccine interferes with diagnosis.
Vaccinated cats have antibodies to FIV which are detected in the
common diagnostic tests. Therefore, it is impossible to distinguish
vaccinated and infected cats in this way.

7. The future: can more effective vaccines be
produced?
• This is not yet clear. A great deal of research is being done by research
laboratories and other pharmaceutical companies to produce FIV vaccines.
• The primary aim is to develop a vaccine that induces both strong cell-
mediated and humoral immunity in order to prevent the establishment of a
persistent infection, which, as for HIV, is difficult to achieve.
• If elimination of the virus cannot be achieved, a vaccine that reduced the
virus load in a subsequently infected cat might still be very useful because
it might reduce the risk of further transmission of the virus, and the
development of disease in the infected cat.
• In future it will be important to carry out FIV vaccine trials to try to protect
against natural infection.

8. Fig : Disease course of FIV and HIV infections. FIV
and HIV infections are characterised by a
progressive decline in the CD4+ T cell subset and
by vigorous humoral and cellular immune
responses that can contain viral replication and
viremia during the asymptomatic phase but
become exhausted in later stages of infection

9. THE SUCCESSES AND FAILURES TOWARD
COMMERCIAL FIV VACCINE
• Massive efforts to develop a prophylactic FIV vaccine began in 1989
after the confirmation of FIV pathogenesis in laboratory cats. In
1990–1998, the first FIV vaccine approaches to be tested were the
inactivated whole-virus (IWV) and inactivated infected whole-cell
(IWC
• But not all FIV strains can serve as vaccine immunogens against
homologous challenges, and even more against heterologous
(i.e.,different from vaccine strain) challenges.
• The efforts to enhance IWC/IWV vaccine immunity consisted of
combining FIV immunogens from multiple subtypes and the use of
new adjuvant formulations

10. Limitations of some current
vaccines types
So, if inactivated and live attenuated FIV vaccines afford protection,
how did they do so??
As regards live attenuated vaccines, resistance to superinfection is likely
to depend not only on antiviral immunity, but also on viral interference
• Moreover, inactivated vaccines are often presumed to be unsuitable
for vaccination against incurable diseases such as AIDS

11. Dual Subtype Fel-O-Vax FIV vaccine
• Perhaps the very nature of the Fel-O-Vax FIV vaccine—based on
based on inactivated whole virus—is in part to blame
• Perhaps the efficacy of the new FIV vaccine has not been sufficiently
well established. Indeed, the vaccine has not afforded protection in
laboratory trials against a highly virulent FIV strain, which may or may
not be typical of naturally encountered viruses.

12. • In 2014, Janet K. Yamamoto
defined an FIV tier system using two related FIV dual-subtype (A+D) vaccines:
the commercially available inactivated infected-cell vaccine (Fel-O-Vax® FIV)
and its prototype vaccine solely composed of inactivated whole viruses.
• Surprisingly, they claimed that Both vaccines afforded combined protection
rates of 100% against subtype-A tier-1 FIVPet, 89% against subtype-B tier-3
FIVFC1, 61% against recombinant subtype-A/B tier-2 FIVBang, 62% against
recombinant subtype-F′/C tier-3 FIVNZ1, and 40% against subtype-A tier-2
FIVUK8 in short-duration (37–41 weeks) studies.
• But
In long-duration (76–80 weeks) studies, the commercial vaccine afforded a
combined protection rate of at least 46% against the tier-2 and tier-3 viruses

13. Surprisingly……….
• The dual-subtype FIV vaccines conferred a statistically significant cross-
subtype protection rate of 89% against a subtype-B virus, which was better
than those against recombinant subtype-A/B and -F′/C viruses.
• Other FIV researchers have also described the efficacy of the Fel-O-Vax®
FIV vaccine, which ranged from high efficacies against the subtype-B
Japanese FIVAO2 , subtype-A FIVFD/US , and subtype-A FIVFD/DutchA
viruses, to no sterilizing efficacy against the subtype-A FIVUK8 virus .
• But, Based also on the work of Dunham et al. the prototype IWV with the
IL-12 supplement was generally more effective against heterologous
isolates than the Fel-O-Vax® FIV. Collectively, these studies suggest the
prototype IWV is better than Fel-O-Vax® FIV. This may be due to the fact
that the prototype IWV consists of only inactivated whole virus, while Fel-
O-Vax® FIV consists of inactivated infected cells

14. Moreover, The prototype (dual-subtype IWVs) and Fel-O-Vax® FIV
(dual-subtype IWVs plus infected cells) vaccines conferred protection
against non-vaccine subtype-B viruses.
However, little is known about the duration, magnitude, and
mechanism(s) of the vaccine protection against other subtype and
recombinant viruses

15. Efficacy criteria for FIV vaccine
• the large genetic diversity of FIV may make it more difficult to derive a
single universal vaccine against the circulating
• FIV subtypes and recombinants. Furthermore, the ability of FIV to be
more polytropic in immune cells than HIV is another problem in the
development of the vaccine
• However, there are key advantages in the development of a FIV
vaccine over an HIV-1 vaccine.
• Another advantage of FIV vaccine development is the natural FIV
transmission mode (via biting), which may be an easier route to
protect than the mucosal transmission route

16. Development of Inhibitors of FIV PR
• 1.The aspartic protease. PR, is responsible for viral Gag and Gag-Pol polyprotein
processing into individual structural and enzymatic proteins during assembly and
maturation.
• Therefore, PR has been a very important target for antiviral therapies
• Several approved protease inhibitors are available that are effective for treating
HIV-1infection and combination drug therapies HAART
• Hence John Elder et al. We have prepared a series of mutant FIVPRs in which HIV-
1 amino acid residues have been substituted into the FIV PR background at
equivalent positions. Confirmation of the involvement of several of these
residues in both substrate and inhibitor specificities has been obtained.
• Structural and biochemical analyses have been performed by that group on FIV
PR to define distinctions with HIV PR to aid in understanding of the basis for
processing

17. FIV protease
• like HIV-1 protease, It is is a homodimeric aspartic proteinase and the
two enzymes
• are very similar at the crystallographic level, particularly within the
substrate binding pocket
• Moreover, Nucleoside analogs that interact with theactive site of
reverse transcriptase have been found efficacious against both FIV
and HIV .
• It is likely that a similar strategy will evolve for development of anti-
integrase drugs.

18. Problem of Antiretroviral Drugs ?
• Although HIV infection has not been eliminated with antiviral therapy,
at such low levels of viral replication, mutations in the viral genome as
greatly slowed and escape from immune recognition and drug
inhibition is limited.
• But, Anti-retroviral therapy is not widely used in FIV-infected cats, and
compounds are rarely designed for molecular interaction with FIV
proteins

19. • The sera from commercial dual-subtype-vaccinated cats had much higher
VNA titers to homologous
vaccine strains (FIVPet and FIVShi) than to either heterologous-subtype-B
strains or homologous-subtype-
• A FIVGL8 (Fig. ) In contrast, sera from prototype dual-subtype-vaccinated
cats had high VNA titers to vaccine strain FIVPet, but had little-to-no VNA
titers to vaccine strain FIVShi

20. Why DNA vaccines against FIV
• DNA Vaccination Affords Significant Protection against Feline
Immunodeficiency Virus Infection without Inducing Detectable
Antiviral Antibodies
• Immunogenicity of a lentiviral-based DNA vaccine driven by the 5′LTR
of the naturally attenuated caprine arthritis encephalitis virus (CAEV)
in mice and macaques
• Ref:

21. Planning to develop DNA vaccine against FIV
• Two Key Attempts…….
1.Deletion of Integrase
The method consists in the deletion of the gene
• the integrase to prevent the integration of the provirus genome.
• To do this, the amplification of parts upstream genes (End of RT) and downstream
(Beginning of Vif) integrase was performed.
• Moreover, this RNA virus integrates into the genome of its host through the integrase
gene. Without integration into the genome, there is no replication. The creation of a
DNA vector lentiviral was initiated during the course.
Indeed, the first strategy for vaccine consists in the deletion of the integrase thereby
preventing integration of the proviral DNA into the host genome. The small amount of viral
genome can be produced and package deletion of integrase possible to avoid having a
persistence of the virus in tissues.

22. Interestingly, the use of CAEV LTR introduces
a novel level of safety
One research group examined whether a chimeric HIV DNA vaccine (CAL-Δ4-
SHIVKU2) whose genome was driven by the LTR of the goat lentivirus, caprine
arthritis encephalitis (CAEV) and expressed efficiently the vaccine antigens and
induces potent immune responses in animal models for HIV vaccine.
So they removed the SIV 5'LTR from the construct and replaced it with the LTR from
lentivirus, CAEV that did not induce AIDS in his host. That LTR was previously
characterized to be Tat-independent therefore to have a constitutive promoter for
gene expression . That novel HIV vaccine was named CAL-Δ4- SHIVKU2.
Moreover, the parental vaccine genome Δ4SHIVKU2, the new non-integrating, non-
replicating vaccine CAL-Δ4-SHIVKU2 expressed efficiently all the antigens encoded
by the vaccine genome in transfected HEK-293T cells.

23. 2.Switching the FIV LTRs with the LTRs of CAEV
The second part is the replacement of LTRs of IVF sequence by those
virus caprine arthritis encephalitis (CAEV). These regions contain information on
the start and on the transcription termination. The LTRs CAEV being active
promoters constitutively, it helps to have the transcript directly without latency.
Reference:

24. • Furthermore it has been demonstrated by other group that the
recombinant IN protein of CAEV did not allow in vitro integration of
HIV LTR DNA .
• From that point of view they hypothesized that a SHIV genome, in
which the SIV LTRs are replaced with those of CAEV, and used as DNA
vaccine, will generate an integration-deficient viral genome but will
retain the capacities to undergo one cycle of replication during which
viral proteins will be expressed from the episomal unintegrated DNA
without its disadvantages namely integration and persistence
associated with the potential of reversion to a pathogenic phenotype.

25. • Cellular culture :CRFK cells (Crandell Feline Kidney) were used during the course
• Trypsinization: to detach the cells to adhereback to the surface by occupying
maximum space to develop
Molecular Experiments
1.Isolation of fragments "End of RT" and "Begining of Vif" parts of upstream and
downstream genes
2.PCR amplification: Fragments "End of RT" and "Begining of Vif" were amplified by
High fidelity PCR of Taq with a gradient program

26. • Bacterial Transformation using E.coli JM109 competent bacteria
Purification of DNA: In order to extract the DNAs present in the gels
Ligation of the fragment "End of RT and Beg of Vif in plasmid genome
of FIV 34 to obtain a plasmid delta IVF integrase

27. Molecular Cloning strategies for 2nd step
• 1. PCR
• Vector restriction digest
• Gel purification
• Insert restriction digest
• Ligation
• Transformation and plating
• Plasmid miniprep
• Depositing clones in a library

28. Concluding remarks
• So…. FIV AIDS model is gaining in momentum and offers unsurpassed
advantages as compared to non-human primate models to devise
novel strategies to vaccinate against lentiviruses using a statistically
meaningful number of animals and with the potential to validate the
approach in the field.
Moreover, The model is very suitable for testing prophylactic and
therapeutic approaches, though experimental work with cats is
ethically challenging and costly, but to a much lesser extent than use of
primates.