Assessment of suitability of two serotype A candidate vaccine strains for inclusion in FMD vaccine in India

Assessment of suitability of two serotype A candidate vaccine

strains for inclusion in FMD vaccine in India

Jajati Keshari Mohapatra a,*, Divakar Hemadri a, T.V.S. Rao b, B.P. Sreenivasa b,Saravanan Subramaniam a, Aniket Sanyal a, Tamilselvan Ramasamy Periyasamy a,

Nawal Kishor Singh a, Bramhadev Pattnaik a, Ramamurthy Venkataramanan b

a Project Directorate on Foot-and-Mouth Disease, Indian Veterinary Research Institute Campus,

Mukteswar-Kumaon, Nainital 263138, Uttarakhand, Indiab Indian Veterinary Research Institute, Bangalore Campus, Hebbal, Bangalore 560024, India

Received 26 November 2007; received in revised form 25 February 2008; accepted 28 February 2008

Abstract

The recent type A foot and mouth disease virus field isolates recovered in India are shown to be antigenically quite divergent

from the in-use vaccine strain (IND 17/82), warranting the selection of a suitable vaccine strain which can cover this diversity in

antigenic spectrum. In earlier studies employing neutralization test with anti-146S rabbit sera raised against eight candidate

vaccine strains, IND 81/00 and IND 40/00 belonging to genotype VII were found to offer the best antigenic coverage. In order to

assess the credibility of IND 81/00 and IND 40/00 as vaccine strains, 17 recent isolates received during 2005–2006 and

representative isolates from older genotypes were subjected to two-dimensional micro-neutralization assay using bovine

convalescent serum (against IND 81/00 and IND 40/00) and bovine vaccinate serum (against IND 40/00). From the results it is

evident that both the isolates IND 81/00 (antigenic relationship ‘r-value’ >0.40 with 86% of isolates) and IND 40/00 (‘r-value’

>0.40 with 78% of isolates) show nearly equal antigenic relatedness with the recent field viruses and hence both of these are

effective vaccine candidates in present context. Though very limited in its extent, these useful data obtained with antisera raised

in homologous host system are logical extension of the on going quest for the appropriate vaccine strain and circumvents species

disparities in the immune recognition of epitopes.

# 2008 Elsevier B.V. All rights reserved.

Keywords: FMD serotype A; Vaccine strain selection

www.elsevier.com/locate/vetmic

Available online at www.sciencedirect.com

Veterinary Microbiology 131 (2008) 65–72

* Corresponding author. Tel.: +91 5942 286004;

fax: +91 5942 286307.

E-mail address: [email protected] (J.K. Mohapatra).

0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2008.02.020

1. Introduction

India is endemic for foot and mouth disease (FMD)

and vaccination has been the sole means to save the

susceptible livestock from this scourge. Of the three

serotypes (O, A and Asia 1) prevalent in India, type A

.

J.K. Mohapatra et al. / Veterinary Microbiology 131 (2008) 65–7266

is antigenically and genetically most heterogeneous.

Four out of 10 global genotypes (Tosh et al., 2002) in

type A have been reported from India and since 2001,

genotype VII has out competed other genotypes and

has been behind all the outbreaks of recent years.

FMD virus is well known for its antigenic volatility

and infamous for giving rise to antigenically and

genetically divergent new strains with time leading to

poor antigenic coverage by in-use vaccine strains

(Brooksby, 1982). This notoriety on part of FMD

virus warrants a constant and elaborate antigenic and

genetic analysis of field viruses in relation to the

vaccine strain for confirming the appropriateness of

existing vaccine strain as well as for selection of new

vaccine strains, if situation demands. Preliminary

serological studies involving type A isolates collected

over a period of 9 years (1993–2001) using two-

dimensional micro-neutralization assay (2D-MNT)

indicated a poor intergenotypic antigenic coverage

among genotype VI and VII isolates (Mittal et al.,

2005; Tosh et al., 2003). To worsen the situation, the

Indian vaccine strain (IND 17/82) belongs to

genotype VI, whereas all the recent outbreaks are

attributed to genotype VII. In such an alarming

scenario, a need for a better vaccine strain that could

offer antigenic coverage to isolates from both the

genotypes was felt and subsequently a search for a

potential type A vaccine strain was initiated (Jangra

et al., 2005). In that study, 39 type A isolates collected

between 1999 and 2003 were subjected to one-way

antigenic analysis against two vaccine strains, viz.

IND 17/77 (genotype IV), IND 17/82 (genotype VI)

and a panel of eight candidate vaccine strains: four

field isolates each representing genotype VI (IND

233/99, IND 237/99, IND 258/99 and IND 68/01) and

genotype VII (IND 6/00, IND 40/00, IND 81/00 and

IND 173/00). Although intergenotypic antigenic

coverage offered by all the candidate vaccine strains

was poor, IND 81/00 and IND 40/00 emerged as better

candidates than all others. However, this extensive

antigenic analysis for selection of vaccine candidates

and their relative efficacy was carried out using anti-

146S rabbit sera. So a logical extension to this work

would be to assess the credibility of IND 81/00 and

IND 40/00 as candidate vaccine strains in homo-

logous target host system as obvious disparities in

epitope recognition pattern by the immune system

existing across species.

In this study, we report the antigenic analysis of

type A FMD field viruses isolated during 2005–2006

and a few selected strains of older genotypes (VI and

IV) in relation to the previously chosen better

candidate vaccine strains, IND 81/00 and IND 40/

00 (both genotype VII) using bovine convalescent sera

(BCS) and bovine vaccinal sera (BVS). Besides, the

study also provides performance of anti-146S rabbit

sera vis-a-vis bovine convalescent serum raised

against IND 81/00 with the field isolates. Finally,

results of the 2D-MNT using anti-146S rabbit sera

against the candidate vaccine strains, IND 258/99

(genotype VI) and the current vaccine strain, IND 17/

82 is also included for comparison purpose.

2. Materials and methods

2.1. Viruses

Seventeen type A FMDV field isolates recovered

during 2005–2006, current vaccine strain IND 17/82,

candidate vaccine strains IND 40/00, IND 81/00, IND

258/99 and a few representative isolates from

genotype IV and VI were used for antigenic analysis.

The history of FMDV field isolates is detailed in

Table 1. The virus isolates obtained from national

repository of FMDV maintained at Project Directorate

on FMD (PD on FMD) were passaged 5–6 times in

BHK-21 cell culture.

2.2. Animal sera

Hyperimmune anti-146S rabbit sera against binary

ethyleneimine (BEI) inactivated cell culture purified

virus particles (IND 258/99, IND 17/82 and IND 81/

00) was obtained from PD on FMD and were produced

as described previously (Tosh et al., 2003). For raising

BVS, IND 40/00 virus was grown in BKH-21 Razi cell

suspension culture and was subsequently purified, BEI

inactivated and oil adjuvanted. Bovines were inocu-

lated with this vaccine at a dose of 8 mg of antigen.

The serum from three vaccinated animals was

collected 21 days postvaccination and pooled. BCS

was raised by injecting intradermolingually 2 � 107

TCID50 dose of virus (IND 81/00 and IND 40/00) in

6–12 months old bovine calf. Serum was collected 21

J.K. Mohapatra et al. / Veterinary Microbiology 131 (2008) 65–72 67

Table 1

History of type A FMD virus field isolates used in this study

PD on FMD designation of virus isolate Date of collection Place of outbreak Host species

IND 17/77 1977 Tamilnadu Bovine

IND 490/97 (17/82)a 1982 West Bengal Bovine

IND 233/99 1999 Hisar, Haryana Bovine

IND 258/99b 12 February 1999 Bhavnagar, Gujarat Bovine

IND 40/00b 29 December 1999 Bangalore, Karnataka Bovine

IND 81/00b 29 January 2000 Bangalore, Karnataka Buffalo

IND 447/05 2005 Guwahati, Assam Not available

IND 22/06 11 November 2005 Kamrup, Assam Bovine

IND 23/06 12 November 2005 Kamrup, Assam Bovine

IND 26/00 2006 Kuchch, Gujarat Bovine

IND 27/06 2006 Kuchch, Gujarat Bovine

IND 28/06 2006 Kuchch, Gujarat Bovine

IND 43/06 15 February 2006 Jammu, J&K Bovine

IND 50/06 22 December 2005 Bangalore Bovine

IND 59/06 19 January 2006 Bangalore Bovine

IND 88/06 9 December 2005 Jabalpur, MP Bovine

IND 97/06 16 February 2006 Gwalior, MP Bovine

IND 99/06 6 March 2006 Madhya Pradesh Bovine

IND 100/06 6 March 2006 Gwalior, MP Bovine

IND 101/06 6 March 2006 Jabalpur, MP Bovine

IND 106/06 2006 Kuchcha, Gujarat Bovine

IND 109/06 26 November 2006 Madhya Pradesh Bovine

IND 152/06 17 March 2006 West Bengal Bovine

a Current vaccine strain.b Candidate vaccine strains.

days postinoculation and pooled from two calves to

avoid obvious individual bias in immune response.

2.3. Neutralization test

In order to estimate the antigenic relationship of the

field isolates, 2D-MNT was performed as per standard

method (Rweyemamu et al., 1978a). In brief, on the

same 96-well plate, seven 10-fold serial dilution of

virus (from 10�1 to 10�7) without serum in duplicate

wells horizontally was tested for determining virus

titre in terms of TCID50 and these dilutions were also

crosschecked by mixing and incubating with five 2-

fold serial dilution of serum (from 1:32 to 1:512) for

determining the dilution of serum that neutralizes 100

TCID50 virus. BHK 21 cells were used as indicator

system in neutralization test and the in vitro titres of

isolates varied from 106 to 108 TCID50/ml. Each test

was repeated thrice on 3 different days and the

averages were considered to avoid manual errors. On

every testing day, along with the isolates the

homologous virus was also tested to avoid any bias.

The end point titre of the serum was calculated as the

reciprocal of the last dilution of serum that neutralizes

100 TCID50 in 50% of the wells. The one-way

antigenic relationship (r-value) was calculated as the

ratio between heterologous and homologous serum

titre (Table 2). The results were interpreted based on

the criteria set by Samuel et al. (1990): an r-value

range of 0.40–1.00 indicates that existing vaccine

strain provide enough protection; while in the range of

0.20–0.39 indicates a need for more potent vaccine.

Conversely, r-value below 0.20 demands for a new

vaccine strain.

2.4. Genetic analysis

For phylogenetic analysis, nucleotide (nt) sequence

of ID region retrieved from local database of PD on

FMD and published previously (Tosh et al., 2002;

Jangra et al., 2005; Mittal et al., 2005; Mohapatra

et al., 2007) were used. Phylogenetic comparison was

performed using MEGA 3.1 software (Kumar et al.,

2004). The Tamura and Nei (1993) model of

J.K. Mohapatra et al. / Veterinary Microbiology 131 (2008) 65–7268

Table 2

One-way antigenic relationship (r-value) of FMDV type A field isolates in 2D-MNT

Genotype PD on FMD

designation of

virus isolate

Bovine

convalescent

serum against

IND 81/00

Bovine

convalescent

serum against

IND 40/00

Bovine

vaccinate

serum against

IND 40/00

Anti-146S

rabbit

serum against

IND 81/00

Anti-146S

rabbit

serum against

IND 258/99

Anti-146S

rabbit

serum against

IND 490/97

(IND 17/82)

IV IND 17/77 0.727 1.00 1.00 <0.2 <0.2 <0.2

VI IND 490/97(17/82) 0.54 <0.2 <0.2 0.35 <0.2 1.00

IND 233/99 0.81 0.85 0.50 0.72 1.00 <0.2

IND 258/99 0.37 0.50 0.33 0.50 1.00 <0.2

VII IND 40/00 1.00 1.00 1.00 1.00 <0.2 0.33

IND 81/00 1.00 0.90 0.87 1.00 <0.2 <0.2

IND 447/05 1.00 0.80 0.75 0.63 <0.2 <0.2

IND 22/06 1.00 1.00 1.00 1.00 <0.2 <0.2

IND 23/06 1.00 1.00 1.00 0.68 <0.2 <0.2

IND 26/06 1.00 1.00 0.75 0.75 <0.2 <0.2

IND 27/06 1.00 0.90 1.00 1.00 <0.2 0.25

IND 28/06 1.00 0.90 0.87 1.00 <0.2 <0.2

IND 43/06 0.72 0.30 0.37 1.00 <0.2 <0.2

IND 50/06 0.68 1.00 1.00 1.00 <0.2 <0.2

IND 59/06 1.00 0.38 0.37 0.38 <0.2 <0.2

IND 88/06 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2

IND 97/06 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2

IND 99/06 0.9 0.90 0.87 1.00 <0.2 <0.2

IND 100/06 1.00 0.94 1.00 1.00 <0.2 <0.2

IND 101/06 0.9 0.75 0.62 1.00 <0.2 <0.2

IND 106/06 0.9 0.90 1.00 1.00 <0.2 <0.2

1ND 109/06 0.54 0.50 0.42 0.75 <0.2 <0.2

IND 152/06 1.00 1.00 1.00 1.00 0.44 <0.2

nucleotide substitution with gamma-distribution

among-site rate heterogeneity (with eight categories)

(termed as TrN + G model) was used to construct the

tree and the tree topologies were evaluated by

bootstrap analysis from 10,000 replicates of the

original dataset. Nucleotide and amino acid (aa)

alignment and divergence estimation was carried out

using ClustalW (Thompson et al., 1994).

3. Results

3.1. Phylogenetic analysis

The phylogenetic tree at 1D region comprising of

23 Indian type A FMDV isolates, including the

vaccine strains (Fig. 1.), depicts three genotypes with

more than 15% nt divergence among them. The

genotype grouping is supported by high bootstrap

confidence. The genotype VII carries the candidate

vaccine strains (IND 81/00 and IND 40/00), where all

recent outbreak isolates are also clustered. But the

current vaccine strain (IND 17/82), though belongs to

genotype VI, lies in a genetically distinct clade

isolated from other members of that genotype. IND

258/99, a candidate vaccine strain of genotype VI

(Jangra et al., 2005) also maintains significant genetic

distance from the current vaccine strain. IND 40/00

and IND 81/00 showed only 0.5% amino acid

divergence between them at VP1 region confirming

their close genetic relatedness.

3.2. Antigenic analysis

As a routine test for deciphering antigenic relation-

ship and for relevant vaccine strain matching exercise,

2D-MNT was applied using anti-146S rabbit sera

against candidate (IND 81/00 and IND 258/99) and

J.K. Mohapatra et al. / Veterinary Microbiology 131 (2008) 65–72 69

Fig. 1. Neighbour joining tree at 1D region showing the phyloge-

netic relationship and genotype classification of FMDV type A

isolates. Bootstrap support value above 70% are shown. Current

vaccine strain is shaded in a box. Candidate vaccine strains and

genotype VI representative isolate are underlined.

in-use vaccine strain (IND 17/82). One-way antigenic

relationship (r-value) of type A isolates against these

strains representing two genotypes are shown in

Table 2. Out of 22 field isolates analyzed, only two

isolates showed an r-value in the range of 0.20–0.39

and rest of them had an r-value of <0.20 with the

current vaccine strain (IND 17/82) indicating its poor

antigenic relationship with recent field isolates

(Fig. 2). Similarly with IND 258/99 representing

genotype VI, only two isolates revealed an r-value of

>0.4. Whilst 77% (n = 17) and 9% (n = 2) of isolates

demonstrated an r-value in the range of 0.40–1.00 and

0.20–0.39, respectively with IND 81/00 anti-146S sera

confirming its wider antigenic relatedness with the

recent isolates.

In order to have a comprehensive antigenic

comparison of genotype VII candidates (IND 81/00

and IND 40/00) in homologous system, serum raised

in target host species for FMD, i.e. BCS (IND 81/00

and IND 40/00) and BVS (IND 40/00) were analyzed

for their neutralizing efficacy for recent isolates and

isolates from other genotypes. Here 20 out of the 22

tested isolates showed an r-value more than 0.2 with

IND 81/00 BCS while 19 of the 22 isolates showed an

r-value in the same range with IND 40/00 BCS and

also BVS (Fig. 2 and Table 2). Thus, from this result it

is evident that both IND 40/00 and IND 81/00 had

almost equal r-value relationships with the recent field

isolates tested.

Interestingly, isolates IND 88/06 and IND 97/06

revealed an r-value of <0.20 with all the sera tested

including IND 81/00 (both rabbit and bovine sera) and

IND 40/00 (both BCS and BVS) indicative of their

exceptional antigenic divergence. At the same time,

IND 109/06 and IND 152/06 which cluster with IND

88/06 and IND 97/06, respectively in the phylogenetic

tree have shown r-value of >0.4 with both IND 81/00

and IND 40/00 sera.

4. Discussion

In an FMD endemic country where vaccination is

the most important means of disease control, it is

imperative that a constant watch on the antigenic

makeup of circulating field viruses and their antigenic

and genetic relationship with the in-use vaccine strains

be maintained for updating vaccine strains. For this, in

vitro neutralization test is considered to be more

relevant to in vivo protection than other measures of

virus–antibody interactions, although non-neutraliz-

ing antibodies may also play critical role in terms of

protection (Dunn et al., 1998; McCullough et al.,

1992).

Here, we present antigenic analysis of 17 recent

field viruses isolated between 2005 and 2006 with

current vaccine strain (IND 17/82) and candidate

strains from two genotypes (IND 81/00 and IND 40/00

from genotype VII; IND 258/99 from genotype VI) in

2D-MNT as a continuation of the search for a better

relevant vaccine strain. As observed earlier (Jangra

et al., 2005), the circulating field viruses are also

antigenically unrelated with the current vaccine strain,

making it irrelevant for inclusion in the vaccine

formulation any more. This is evident from the ‘r-

value’ pattern observed for the recent isolates with

IND 17/82 (Fig. 2), where 94% of the isolates tested

showed an r-value of <0.2. The antigenic relatedness

J.K. Mohapatra et al. / Veterinary Microbiology 131 (2008) 65–7270

Fig. 2. Antigenic relationship (r-value) of type A field isolates with current and candidate vaccine strains as established in 2D-MNT.

of the recent isolates with IND 258/99, a candidate

strain from genotype VI, was also observed to be very

poor, where except one isolate all others showed an r-

value of <0.2. This is in corroboration with the earlier

trend where a poor intergenotypic antigenic coverage

between genotype VI and VII isolates (Mittal et al.,

2005; Tosh et al., 2003) was demonstrated. Although

this strain has been established to offer a superior

antigenic coverage to isolates of its own genotype VI

(Jangra et al., 2005), the present situation where since

2001, studied outbreaks have been exclusively due to

genotype VII (unpublished observation, PD on FMD),

makes it an unsuitable candidate for vaccine

formulation. Out of the panel of four strains belonging

to genotype VII, IND 81/00 followed by IND 40/00

have proved their antigenic superiority in an earlier

analysis using anti-146S rabbit sera (Jangra et al.,

2005). As discrimination in immune recognition of

epitopes exist across species, the antigenic analysis of

the field isolates was carried out using bovine sera for

proper resolution of antigenic relatedness.

Using BCS, 19 of the 22 isolates and 17 of the 22

showed an r-value of >0.40 with IND 81/00 and IND

40/00, respectively indicating that both these isolates

are able to offer better antigenic coverage. Inter-

genotypic coverage of IND 81/00 (BCS) was found to

be slightly better than IND 40/00 (BVS), as one of the

three isolates of genotype VI had an r-value of <0.20

with IND 40/00, but none of them showed an r-value

of <0.20 with IND 81/00. Though number of isolates

from genotype VI under study is less, this observation

is in compliance with the earlier one made by Jangra

et al. (2005), where no significant difference was

observed in intergenotypic coverage of IND 81/00 and

IND 40/00 using anti-146S rabbit sera. Even if IND

81/00 and IND 40/00 did not show perfect antigenic

relatedness with all the isolates of genotype VII under

study, being strains which cover the diversity of

circulating type A FMDV to the maximum extent and

for the fact that genotype VI is no more reported in

field outbreaks in recent times, strengthens their

candidature as future vaccine strains. Further, these

genetically related strains have shown only one amino

acid difference at VP1 region between them and have

been collected from outbreaks that occurred just 1

month apart in the same place. Therefore either of

these strains of genotype VII may be considered for

vaccine inclusion based on their near equal antigenic

relatedness with circulating field viruses. The isolate,

IND 40/00 has already shown to adapt and grow well

J.K. Mohapatra et al. / Veterinary Microbiology 131 (2008) 65–72 71

in suspension cells for vaccine production (results not

shown) but the growth characteristics of IND 81/00

remains to be assessed. Besides, anti-146S rabbit

serum against IND 81/00 revealed an r-value of <0.2

with genotype IV isolate (IND 17/77), whereas BCS

showed an r-value of >0.40. This again supports the

species-specific immune recognition pattern and the

rationale behind determining antigenic relationship

using serum raised in target host system for flawless

interpretation.

Usually vaccine matching tests employ BVS raised

against vaccine strains (Paton et al., 2005), partly

because its production does not require handling of

live virus outside the laboratory and partly because of

the variability in result in terms of virus specificity

reported for convalescent sera (Rweyemamu et al.,

1978b). But in this study, the serum against IND 81/00

is a convalescent serum whereas against IND 40/00

both convalescent and vaccinal sera were applied. As

it is established that BEI inactivation can compromise

the integrity of certain proteins of the virus

(Kasermann et al., 2001), which may lead to loss of

certain immunologically critical neutralizing epitopes,

comparison of results obtained with BVS and that with

BCS might be questionable. Notably, comparison of

‘r’ values obtained from convalescent and vaccinal

sera against IND 40/00 shows similar performances by

both these sera indicating use of convalescent sera did

not affect the final inference of the test results in our

study. Still to elucidate how IND 81/00 behaves after

BEI inactivation and for a better comparison with IND

40/00, BVS against IND 81/00 remains to be produced

and tested with all circulating field viruses.

To sum up, in compliance with the earlier findings,

both the isolates IND 40/00 and IND 81/00 are found

equally better candidate vaccine strains for replace-

ment of the existing vaccine strain. The current

vaccine strain, IND 17/82 and the candidate vaccine

strain from genotype VI (IND 258/99) continues to

offer poor intergenotypic coverage to recent isolates

belonging to genotype VII. Hence in present scenario,

where genotype VII has been exclusively associated

with all the field outbreaks in India since more than 6

years, IND 81/00 and IND 40/00 by virtue of their

superiority in their antigenic and genetic relatedness to

the circulating field isolates emerge to be the candidate

vaccine strains of choice. Not withstanding this fact,

complete genomic characterization of these two

strains and antigenic matching with more number of

circulating field viruses need to be undertaken.

Acknowledgements

We are thankful to Indian Council of Agricultural

Research for providing necessary facilities to carry out

this work. Technical assistance of Shri N.S. Singh and

Shri G. Singh, PD on FMD and Shri H.R. Narayana,

TO of FMD vaccine production at IVRI, Bangalore

campus is appreciated.

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