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Vaccine 27S (2009) F130–F138

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

evelopment and characterization of candidate rotavirus vaccine strains derivedrom children with diarrhoea in Vietnam

uan T. Lea,∗, Trang V. Nguyenb, Phuong M. Nguyenb, Nguyen T. Huonga, Ngo T. Huonga,guyen T.M. Huonga, Tran B. Hanha, Dang N. Haa, Dang D. Anhb, Jon R. Gentschc, Yuhuan Wangc,athew D. Esonac, Roger I. Glassd, A. Duncan Steelee, Paul E. Kilgore f, Nguyen V. Mana,

aoming Jiangc,∗∗, Nguyen D. Hiena

Center for Research and Production of Vaccines and Biologicals (POLYVAC), 135 Loduc Street, Hai Ba Trung, Hanoi, Viet NamNational Institute of Hygiene and Epidemiology, Hanoi, Viet NamDivision of Viral Diseases, U.S. Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USAFogarty International Center, National Institutes of Health, Bethesda, MD, USAVaccines and Immunization, PATH, Seattle, WA, USAInternational Vaccine Institute, Seoul, Republic of Korea

r t i c l e i n f o

eywords:otavirus vaccineiarrhoeaietnam

a b s t r a c t

In Vietnam, rotavirus infection accounts for more than one-half of all hospitalizations for diarrhoea amongchildren less than 5 years of age. While new vaccines to prevent rotavirus diarrhoea have been developedand introduced into some countries by multinational manufacturers, the ability for developing countriessuch as Vietnam to introduce several new and important vaccines into the routine infant immunizationschedule may be challenging. In order to be partially self-sufficient in vaccine production, Vietnam haspursued the development of several rotavirus strains as candidate vaccines using isolates obtained from

Vietnamese children with diarrhoea. This paper describes the origin, isolation and characterization of3 human rotavirus strains being considered for further vaccine development in Vietnam. The goal isto prepare a monovalent G1P [8] rotavirus vaccine using one of these strains obtained in Vietnam andnaturally attenuated by multiple passages in cell culture. While this is an ambitious project that willrequire several years’ work, we are using the lessons learned to improve the overall quality of vaccineproduction including the use of Vero cell techniques for the manufacture of other vaccines in Vietnam.

. Introduction

From 1998 to 2003, we conducted sentinel hospital surveil-

ance of rotavirus diarrhoea among children less than 5 years ofge seen in four cities of Vietnam. Rotavirus was identified inore than 50% of children hospitalized for diarrhoea [1,2], indi-

ating a strong need for a vaccine that could substantially reduce

Abbreviations: US CDC, United States Centers for Disease Control and Preven-ion; EIA, enzyme immunoassay; GCP, good clinical practice; GLP, good laboratoryractice; GMP, good manufacturing practice; MS, master seed; PMKC, primaryonkey kidney cells; PRKC, primary rabbit kidney cells; MA104, African greenonkey kidney cells; PAGE, polyacrylamide gel electrophoresis; PL, passage level;

OLYVAC, Center for Research and Production of Vaccines and Biologicals, Hanoi,ietnam; RT-PCR, reverse transcription-polymerase chain reaction; WHO, Worldealth Organisation; WS, working seed.∗ Corresponding author. Tel.: +84 48213820; fax: +84 48213203.

∗∗ Corresponding author. Tel.: +1 4046392861; fax +1 4046393645.E-mail addresses: luanpolyvac@gmail.com (L.T. Le), bxj4@cdc.gov (B. Jiang).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.08.086

© 2009 Elsevier Ltd. All rights reserved.

severe rotavirus hospital admissions, outpatient visits, and asso-ciated deaths. In an analysis of rotavirus strains identified fromprospective surveillance, G1P[8] was the most commonly identifiedstrain (45%). The other common rotavirus strains—G2P[4], G4P[8],G4P[6] and G9P[8], were also present, and a few rare strains werefound (e.g., G1P[4] and G9P[4]).

Vietnam has a strong history of self sufficiency in localmanufacturing of vaccines. While many new vaccines are becom-ing available for immunization of children, the government isconcerned about the affordability of these products and has encour-aged the domestic manufacture of many new vaccines. In recentyears, the government, with international partners, has devel-oped new manufacturing capacity for vaccines against HepatitisB, Japanese B encephalitis, and measles [3,4], and based on the high

burden of rotavirus disease, has recently provided seed funding todevelop rotavirus vaccine.

The demonstration that a live, oral rotavirus vaccine could beproduced from a single human strain, attenuated by multiple pas-sages, and manufactured in Vero cells, encouraged us to apply a

L.T. Le et al. / Vaccine 27S (2009) F130–F138 F131

Table 1Adaptation and passage history of the 3 candidate human rotavirus vaccines for use in Vietnam.

Strain KH 0118 SP 210-2000 SP 203-2001Origin 6-month-old girl in Khanh Hoa

Hospital, Nha Trang, 200311-month-old boy in Saint PaulHospital, Hanoi, 2000

9-month-old boy in Saint PaulHospital, Hanoi, 2001

Genotype G1P[8] G1P[4] G4P[6]

Passage history Stool Stool Stool↓ ↓ ↓Isolation in MA104 cells (3×) Isolation in MA104 cells (3×) Isolation in MA104 cells (3×)↓ ↓ ↓Plaque purification in MA104 cells (1×) Plaque purification (3×) in MA104 cells Plaque purification (2×) in MA104 cells↓ ↓ ↓6 Passages in MA104 cells 9 Passages in MA104 cells 6 Passages in MA104 cells↓ ↓ ↓12 Passages in PMKC* 11 Passages in PMKC 24 Passages in PMKC↓ ↓ ↓18 Passages in Vero cells 10 Passages in Vero cells 12 Passages in Vero cells↓ ↓ ↓Cloning by limiting dilution 2 times in Vero cells Cloning by limiting dilution 2 times in Vero cells Cloning by limiting dilution 3 times in Vero cells↓ ↓ ↓4 Passages in Vero 4 Passages in Vero 4 Passages in Vero↓ ↓ ↓

* seed (

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Master virus seed (PL5) Master virus↓ ↓Working virus seed (PL6) Working viru

ote: *PMKC: primary monkey kidney cells; PL: passage level.

imilar strategy in Vietnam since these methods are already used inur existing vaccine production activities [5]. In addition, in the pro-ess of developing a rotavirus vaccine, we have been able to updateome methods and procedures used in the manufacturing of otheraccines. Finally, by adapting a local rotavirus strain with severaldditional strains as backup candidate rotavirus vaccine strains,e will be better able to match vaccine viruses to local rotavirus

trains in circulation and thus, achieve an equivalent or higher levelf protection compared with currently available rotavirus vaccines.

. Materials and methods

.1. Overall strategy

Our strategy to develop a live, oral rotavirus vaccine forietnam has involved the selection of the most common circulat-

ng serotype, G1P[8] recovered from an 6-month-old child withotavirus diarrhoea captured in the national surveillance programlong with two other strains (G1P[4] and G4P[6]) detected inoung infants. These strains were adapted and passaged in cellulture to select the isolates that grew best and to highest titren Vero cells. The strains were also fully characterized, and pro-eeded through at least 40 blind passages with the assumption thatatural attenuation would occur over time. Following testing ofhe candidate vaccine strains which included toxicity testing andnimal studies, our next steps include human studies: to ensurehe safety of the strains, to determine if they have been ade-uately attenuated for infants, and to ensure that they still induce aobust immune-response. This paper describes the selection, pas-aging, initial characterization and preliminary safety testing ofur selected strains. For guidelines, we followed the WHO pub-ished procedures and methods to develop oral rotavirus vaccines6]. All work was conducted in a special laboratory at the Centersor Disease Control and Prevention (CDC) in Atlanta and POLYVAC,facility approved by national authorities in Vietnam for the pro-uction of vaccines for polio and measles.

.2. Origin and properties of Vietnam human rotavirus vaccineandidates

Three faecal specimens from otherwise healthy Vietnamesenfants with acute diarrhoea, that tested positive for rotavirus

PL5) Master virus seed (PL5)↓

(PL6) Working virus seed (PL6)

by EIA and PCR and contained representative genotypes G1P[8],G1P[4], and G4P[6] of rotavirus, were selected for further develop-ment (Table 1) [7,8]. These strains were first adapted to grow inMA104 cells, a cell line derived from monkey kidney cells. To avoidconcerns about contamination with the agent of bovine spongi-form encephalopathy, we used MA104 cells that were originallyreceived in January 1979 at passage 14 from MA Bioproducts whichhas changed to Bio Whittaker (Walkersville, MD) and had beenstored at the Cell Culture Laboratory, Biologics Branch, US CDC.We have complete passage history and full documentation forthese MA104 cells. Rotavirus strains were purified and serially pas-saged in MA104 cells and primary monkey kidney cells (PMKC) toincrease the rotavirus virus titres. A final stage included passage inVero cells qualified for vaccine production of a master and work-ing virus seed. The PMKC were drawn from the production facilityfor OPV which is operated by POLYVAC and has previously under-gone rigorous routine procedures for safety testing as requiredfor OPV vaccine production under Vietnamese national regulatoryprocedures.

The human rotavirus (HRV) strain G1P[8] (KH0118) wasobtained from a 6-month-old girl in Khanh Hoa General Hospital(Nha Trang, Vietnam) in 2003. This is the most commonly occur-ring strain globally and also in Vietnam and proof of principle existswith the monovalent Rotarix® vaccine [5]. This virus was isolatedby passaging 3 times and plaque-purified once in MA104 cells. Itwas further passaged 6 additional times in MA104 cells and then 12additional times in PMKC. The virus was then adapted and passaged18 times in Vero cells that were qualified for use in vaccine produc-tion. Individual clones were selected by limiting dilution twice inVero cells. After four more passages in Vero cells, a master seed(MS) was produced at passage level (PL) 5. One additional passageof MS in Vero cells produced a working seed (WS) of PL 6 for a totalpassage number of 48.

The HRV strain G1P[4] (SP210-2000) was derived from an 11-month-old boy in Saint Paul Hospital (Hanoi, Vietnam) in 2000. Thevirus was isolated, plaque-purified 3 times and passaged 9 more

times in MA104 cells (Table 1). The virus was then passaged 11times in PMKC and adapted to grow in Vero cells (10 passages). Aftercloning by 2 rounds of limiting dilution, the virus was amplified inVero cells to prepare MS (PL5) and WS (PL6) for a total passagenumber of 44.

F e 27S (2009) F130–F138

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Fig. 1. RNA profiles of rotavirus vaccine candidates, G1P[8], G1P[4] and G4P[6] iso-lated in Vietnam. Rotavirus dsRNA was extracted from infected Vero cell cultures

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132 L.T. Le et al. / Vaccin

A G4P[6] strain (SP203-2001) was identified from a 9-month-ld boy in Saint Paul Hospital in 2001. The virus was isolated bynitial passage and plaque purification twice, and passaged 6 timesll in MA104 cells. After 24 passages in PMKC, the virus was adaptedo grow in Vero cells (12 passages). After 3 rounds of purificationy limiting dilution, the virus was amplified in Vero cells to prepareS (PL5) and WS (PL6) for a total passage number of 56.

.3. Quality control of master and working rotavirus seeds

The MS and WS for each virus were tested for adventitiousgents by using an array of in vitro and in vivo assays as suggested inhe WHO guidelines for the production of an attenuated rotavirusaccine [6]. The tests used were approved by the national regula-ory authority in Vietnam. The MS and WS were routinely checkedor identity by 10% PAGE (Fig. 1) and by RT-PCR (G and P typing)o assure the correct electropherotypes and G and P genotypes,espectively using published methods [7,8]. Additional tests wereerformed on the virus seeds to confirm the stability of the viralenome and absence of adventitious agents over a defined passageange. The passage level used to qualify the virus seeds is generally–10 passages beyond that used for vaccine production [6]. Ourorking virus seed (PL6) is just one more passage of the master

irus seed (PL5) in Vero cells.

.4. Safety tests

Each master or working seed lot was tested for toxicity, safetynd immunogenicity in animals including guinea pigs, mice, rab-its, and monkeys (Table 2). They were also tested by RT-PCR fordventitious viruses, including foamy virus, hepatitis viruses andycoplasma, by the Research and Development Department at

he Company for Vaccine and Biological Production No1 in Hanoi9–11]. Safety tests for adventitious viruses were also conductedn cell cultures including PMKC, neonatal rabbit kidney cells, Verond Hep 2 cell lines as recommended by the WHO Guidelines [6].

.5. Analysis of NSP4, VP6, VP7 and VP4 genes of candidateaccines

The MS of the 3 candidate vaccines was subjected to sequencenalysis for rotavirus genes 4, 6, 9 and 10, which encode the twoeutralization antigens, the group antigen and the putative viral

nterotoxin. Partial nucleotide sequences of genes 4 and 9 (encod-ng VP4 and VP7, respectively) were obtained from ampliconsenerated in the first round of G and P typing RT-PCR.

Phylogenetic analysis was performed on the deduced amino acidequences from aa 1-175 for NSP4, 1-397 for VP6, 1-288 for VP7 and

able 2ualification tests used for master and working rotavirus seeds and results.

Qualification tests Methods

Hepatitis A virus PCRHepatitis B virus PCRHepatitis C virus PCRSimian virus SV40 PCRSimian foamy virus PCRMycoplasma PCRBacteria and fungi Bacterial and fungal cultuM. tuberculosis Mycobacterial cultureTissue culture safety: Vero, Hep2, PMKC, PRKC* Cytopathic effect, hemedAnimal safety: adult mice, rabbits, Guinea pigs ObservationSafety and immune-response in young monkeys

(6–12-month-old)Observation

Measurement of neutralizby plaque reduction assay

Note: PCR: polymerase chain reaction; PMKC: primary monkey kidney cells; PRKC: prim

and analyzed on 10% polyacrylamide gel, followed by staining with silver nitrate.Lanes 1, DNA marker; 2, G1P[8] vaccine seed; 3, G1P[4] vaccine seed; 4, G4P[6]vaccine seed, and 5, reference Wa strain.

16-278 for VP4. Sequence data were first analyzed using CHROMASsoftware (Technelysium Pty Ltd, Tewantin, Australia). Forward and

reverse sequence data of each sample were aligned using the EditSeq and MegAlign program (DNASTAR Inc., Software, Madison, WI)to obtain the final consensus sequence. Complete alignment wasperformed with Cluster W (DNASTAR Inc., Software, Madison, WI).

Results

NegativeNegativeNegativeNegativeNegativeNegative

re NegativeNegative

sorption NegativeIncrease in weight and appear healthyIncrease in weight and appear healthy

ing antibodys

3-fold increase in serum antibody titres from pre- topost-third dose vaccination

ary rabbit kidney cells.

L.T. Le et al. / Vaccine 27S (2009) F130–F138 F133

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ig. 2. Phylogenetic tree of the NSP4 gene fragments of the 3 Vietnam candidate voxes. The 738 bp PCR product of NSP4 gene was amplified using Jrg30 and Jrg31 prU (Australia), BL (Bangladesh), CN (China), ID (India), JP (Japan), TL (Thailand), TW

. Results

.1. Preparation of candidate rotavirus vaccines

Three rotavirus strains (rotavirus genotypes G1P[8], G1P[4] and4P[6]) from patients enrolled in the surveillance study wereelected as candidate strains for rotavirus vaccine development.

ll 3 patients had diarrhoea with different degrees of dehydrationut no apparent fever. The patients were otherwise in good health.e first adapted rotavirus strains to grow in MA104 cells that were

repared before 1980 with complete detailed documentation andhen passaged the strains in PMKC and qualified Vero cells using

s. The Vietnamese strains used to develop vaccine candidates are indicated in theair and directly sequenced. Abbreviations of countries of isolation in strain names:an) and US (the United States of America).

certified reagents under stringent GLP conditions (Table 1). Strainswere purified by plaque assay and limiting dilution. MS and WSbanks were prepared with Vero cells in roller bottles.

3.2. Preclinical safety and immunogenicity of candidate rotavirusvaccines

The MS and WS of the 3 candidate rotavirus vaccine strains wereshown to be free of extraneous agents (hepatitis A, B and C viruses,SV40, foamy virus, mycoplasma, bacteria and fungi) and alsoappeared to be safe in tissue culture, laboratory animals includingmonkeys (Table 2). All 3 vaccines elicited a 3-fold increase in serum

F134 L.T. Le et al. / Vaccine 27S (2009) F130–F138

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ig. 3. Phylogenetic tree of the VP6 gene fragments of the 3 Vietnam candidate vaccene (1356 bp) was amplified using Jrg7 and Jrg8 primer pair and directly sequencU (Australia), BL (Bangladesh), CN (China), JP (Japan), ID (India), NZ (New Zealand)

eutralizing antibody titres following immunization with 3 vaccineoses compared to pre-vaccination titres in baby monkeys (dataot shown). All 3 strains had long RNA electropherotypes (Fig. 1).f note, the profile of the G1P[4] strain demonstrated a high level of

imilarity to that of reference G1P[8] strain Wa. The G and P types ofhese candidate vaccines were confirmed by RT-PCR and sequencenalysis.

.3. Strain characterization—sequencing

.3.1. NSP4 typingThe NSP4 sequences of 3 candidate rotavirus vaccine strains

ere compared with 175 aa sequence fragments of viruses iso-

ated from Bangladesh, India, Thailand and China in the sameeriod and with reference strains Wa, DS-1, ST-3 and M37. NSP4ene of the KH0118 (G1P[8]) and SP203-2001 (G4P[6]) strainselong to genogroup B (prototypes Wa, ST-3 and M37), whereashe SP210-2000 (G1P[4]) strain belongs to genogroup A (prototype

he Vietnamese vaccine candidates are indicated in the boxes. The full length of VP6ng PCR and internal primers. Abbreviation of countries of isolation in strain name:(Philippines), TL (Thailand) and US (The United States of America).

DS-1) (Fig. 2). We observed several notable differences in the NSP4sequence between the 3 Vietnamese strains in this study, the refer-ence G1P[8] strain, and strains from other countries. In particular,there were substitutions of Thr to Met at aa45, Ile to Val at aa76,Tyr to Cys at aa85, and Ser/Lys for Iso at aa97 (data not shown). Theposition aa131 in the NSP4 toxic peptide (aa114-135) was Tyr forG1P[4] and His for G4P[6] and G1P[8] strains, consistent with theability of the original strains of these vaccine candidates to causediarrhoea in children. However, all of these strains have the Ile at aa135, a marker for attenuation [12], indicating that the strain mighthave been attenuated after 40 or more passages in cell culture. Thesingle strain from Vietnam (KH210-2004) whose NSP4 sequencewas previously available also showed high aa sequence similarity

to our G1P[8] strain and came from the same province in Vietnam.

3.3.2. VP6 typingThe 3 Vietnamese vaccine candidates belong to two major VP6

genogroups. The region of aa280-350 defines VP6 genogroups [13]

L.T. Le et al. / Vaccine 27S (2009) F130–F138 F135

F [4] (4ai rs andi , ID (IA

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ig. 4. Phylogenetic tree of the VP7 gene fragments of the Vietnam G1P[8] and G1Pn the box. The 896 bp PCR products were amplified using 9con1L and VP7R primesolation in strain names: Au (Australia), BL (Bangladesh), BRZ (Brazil), CN (China)merica) and VN (Vietnam).

nd residues RPPN at aa 296-299 are present in all 3 Vietnamtrains, suggesting that they belong in subgroup I or II (Fig. 3). The1P[8] and G4P[6] strains showed characteristics of genogroup II

Asn at aa 305, Asn at aa 339, Leu at aa 342 and Ala at aa 348),

hile the G1P[4] showed characteristics of genogroup I and there-

ore of subgroup I (Ala at aa 305, Asn at aa 310, Glu at aa 315,er at aa 339, Met at aa 342 and Ser at aa 348). Phylogenetically,he Vietnam G1P[4] was in the same cluster as DS-1, SA11, 1076nd AU-1, whereas the Vietnam G1P[8] and G4P[6] strains were in

) and G4P[6] (4b) candidate vaccines. Vietnamese vaccine candidates are indicateddirectly sequenced using PCR and internal primers. Abbreviations of countries of

ndia), ISR (Israel), JP (Japan), TL (Thailand), TW (Taiwan), US (The United States of

the same cluster with Wa, RV3 and ST-3, confirming the classifica-tion of our vaccine strains (Fig. 3). The sequence of VP6 from ourG1P8 strain was similar to those from Bangladesh/2002–2003 andThailand/2001 whereas the sequence of G1P[4] strain was closely

related to those in Bangladesh/2000 and India during the sameperiod and to DS-1 and SA11 (Fig. 3). In addition, the Ala at aa172of the Vietnam G1P[4], DS-1 and SA11 (subgroup I) was distinctlydifferent from the Met at this position for many other subgroup IIstrains including the Vietnam G1P[8], G4P[6], Wa, RV3 and ST-3.

F136 L.T. Le et al. / Vaccine 27S (2009) F130–F138

Fig. 5. Phylogenetic tree of the VP4 gene fragments of the 3 Vietnam candidate vaccines. The Vietnamese vaccine candidates are indicated in the box. The 876 bp PCR productw d inteB ThailaV

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as generated using primer pair con3 and con2 and directly sequenced using PCR anL (Bangladesh), CN (China), HGR (Hungary), ID (India), JP (Japan), Korea (KOR), TL (N (Vietnam).

he Vietnam G1P[4] strain also showed amino acid substitutions ofnknown importance at positions aa330 (Val-Ile), aa369 (Glu-Asp)nd aa396 (Ile-Val) compared with other strains.

.3.3. VP7 sequences

The VP7 sequences of the Vietnam G1 candidate vaccine strains

ere similar to other Vietnamese strains (G1-lineage III) whereas

he VP7 sequence of the G4 strain was similar to the correspondingene of other strains from Vietnam, China, Japan, and Thailand andhe porcine Gottfield strain (Fig. 4). The VP7 sequence fragmentsf the two G1 strains were similar to those isolated elsewhere in

rnal primers. Abbreviations of countries of isolation in strain names: Au (Australia),nd), UK (United Kingdom), US (The United States of America), VZL (Venezuela) and

Vietnam during the same period (2003), and similar to those fromBangladesh and Thailand, clustered into lineage III, but differentfrom those isolated from Japan and China or Finland (other lineagesI, II and IV) (Fig. 4a). Within the same lineage III, the Vietnam G1P[4]and G1P[8] were quite different.

3.3.4. VP4 sequenceThe amino acid sequence of VP4 from the Vietnamese G1P[8]

vaccine candidate strain demonstrated that strain KH0118 (G1P[8])falls in the P[8]-2 F45-like lineage, having a high degree of homol-ogy with OP354 strain P[8]-3 (Fig. 5). Many of the Vietnamese

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1P[8], G4P[8] and G9P[8] strains were in the P[8]-2 lineage, andome G3P[8] and G9P[8] are of P[8]-3 lineage [14]. Due to differ-nces in the region sequenced between the VP4 sequences in ourtudy and the previous study, these strains could not be includedn the phylogenetic tree. Amino acid alignments within the sameegion revealed the conserved Pro at aa224 and 225, like many other[8] rotavirus strains. Important substitutions in VP4 sequence ofhe Vietnam G1P[8] included Arg-Thr (aa144) and Arg-Gly (aa268)data not shown).

The VP4 sequence of the P[4] strain was similar to other Viet-amese G2P[4] strains isolated elsewhere. The G1P[4] strain had

mportant substitutions in the VP4 sequence compared to othertrains including changes of Thr-Ala at aa103 and Arg-Gly at aa162.he Vietnam G1P[4] strain shared 96.5–98.1% homology with TB-hen 2004 and Kmr029-2001 strains in the same cluster, and2.6% and 95.3% homology with DS-1 and RV5 of different clusters,espectively (Fig. 5). By contrast, the VP4 sequence of the G4P[6]train shows high similarity to porcine strains. The VP4 sequencesf P6 strains from Vietnam are alike and clustered into a separateineage with those reference strains isolated in 1980s (RV3, ST-3nd M37) and strains from Taiwan and Bangladesh. The G4P6 clus-ers in the same lineage as the VN904 (G9P[6]), VN846 and VN592both are G4P[6] strains). This lineage was identified as P6-Ic, whichas more than 95% similarity to porcine strains 221/04 and 134/0414]. The Vietnam G4P[6] did not have the same conserved residuess other P6 strains at aa 90 and 91, Pro-Ile instead of Qln-Val, aa98rg instead of Lys, aa101 Val instead of Ile, aa140 Tyr instead of Phe,a170-172 Gly-Gly-Arg instead of Tyr-Asn-Ser, aa202 Thr instead ofal, aa232 Val instead of Ile, aa242 Ala instead of Val, and aa255 Val

nstead of Ile. The Vietnam G4P[6] strain exhibited many substitu-ions at the VP4 gene compared to other reference and field strainsn the Asian region. Thus, our G4P[6] strain might have porcinerigins.

. Discussion

This is the first report to describe the development of candidateotavirus vaccines from human strains G1P[8], G1P[4] and G4P[6]solated in Vietnam. These strains were adapted for growth, cloned,nd prepared as MS and WS banks in Vero cells. The G1P[8] strainould potentially be used as a monovalent vaccine with a muchower production cost and a viral sequence that fits better with theirculating P[8] lineage in Vietnam. The other two strains (G1P[4]nd G4P[6]) may be included in a vaccine formulation, if needed,o improve cross-protection and enhance overall vaccine efficacy.urther studies will be needed to determine whether the G1P[4]accine strain induces immunologic cross-protection against both1 and G2 viruses, derived from different rotavirus genogroups.dditional evaluation of the candidate rotavirus vaccine strains iseeded to assess whether or not the multiple passages in cell cul-ure led to attenuation of the virus strain, something that can nownly be determined in human studies.

We have characterized the 3 HRV strains that have been usedor the development of candidate rotavirus MS G1P[8], G1P[4] and4P[6]. These strains represented the major P and G genotypes

G1) that have been circulating in Vietnam [2]. Results of surveil-ance in Vietnam indicate that a P[8] strain can cover over 70% ofhe serotypes in circulation [2]. We also developed P[4] and P[6]accine candidates to cover other major P types (14.4% and 7.6%,espectively) of rotavirus strains circulating in Vietnam. The 3 virus

eeds were biologically cloned by limiting dilution in Vero cells tonhance genetic homogeneity as well as to remove potential adven-itious agents that may have been present in the initial virus inocular derived from the attenuation process [5,6]. The virus seeds werexamined by performing a variety of tests and were qualified to be

(2009) F130–F138 F137

free of adventitious agents [6]. The vaccine candidates were alsodemonstrated to be free of adventitious agents in cell culture andin experimental animal models. All 3 vaccine candidates inducedneutralizing antibody response in young monkeys.

Additional sequence analysis of the NSP4 and VP6 genes of thestrains indicates that our vaccine candidates covered the 2 majorNSP4 genotypes and the 2 subgroups of VP6. Sequence of the NSP4toxic peptide identified one of the markers for attenuation (Ile ataa135), probably acquired during repeated passages in the cells, yetstill showed Tyr/His at aa131, characteristic of the original strainscausing diarrhoea [12]. The VP7 and VP4 sequences of the G1P[8]strain shared similarity to some strains from Vietnam, Bangladesh,Thailand, whose sequences are available for analysis [14]. Analysisof the VP4 sequence indicates that the Vietnamese P8 strain belongsto cluster F45-like (P[8]-2), whereas Rotarix®, one of the currentlylicensed and available rotavirus vaccines, is P[8]-1 [14]. The find-ings that most strains in Vietnam fall into P[8]-2 and P[8]-3 suggestthat it may be important to develop a rotavirus vaccine from localstrains.

Several lines of rationale have led us to consider developing anew vaccine for Vietnam. First, the long-term costs associated withintroduction of several new vaccines may be unsustainable for adeveloping country such as Vietnam and the tradition of being self-sufficient in vaccine production has led the government to investheavily in vaccine production and to support this developmentactivity. A single strain of rotavirus prepared as a vaccine throughmultiple passaging and preparation in Vero cells requires a levelof technology similar to the production of OPV. This productiontechnology has been developed at institutions within Vietnam andhas been adapted for use in development of the rotavirus vaccinecandidates reported here.

Of note, the UK vaccine prepared from bovine reassortant strainshas been licensed by US NIH to emerging manufacturers in eightcountries and could be a direct and less expensive competitor vac-cine to the Merck vaccine, RotaTeq®. This vaccine is much morecomplicated to prepare as it contains multiple strains that need tobe formulated together. The epidemiology of rotavirus in Vietnamis more similar to the epidemiology in a middle income countryrather than a poor developing country. Therefore, we hope that ourcandidate G1 strains might perform as well in Vietnam as the mono-valent GSK vaccine performs in Latin America and South Africa [5].Our vaccine strains have a sequence more homologous to otherstrains in Vietnam than strains elsewhere, including the G1P[8]strain in the GSK vaccine. Therefore, these strains might well pro-vide a slightly better level of protection than those included incurrently available vaccines, a speculation that remains to be exam-ined independently and after a long period of clinical development.

The steps to make a new vaccine are complicated, costly, timeconsuming and fraught with many challenges. This project hasresulted in 3 vaccine candidates that can move into human trials—akey step of the developmental sequence for a rotavirus vaccinethat will take several years to accomplish. To be compliant withWHO and international standards, we need to consider and developprocesses that ensure safe, consistent, robust vaccine productionaccording to GMP practices. In addition, safety, immunogenicityand efficacy in human subjects need to be performed according toGCP. Finally, this process needs to take into consideration the over-all costs and ability to produce this vaccine on a manufacturingscale. All of these various activities will be critical for the ultimatesuccess of the project.

The level of international collaboration and technical support

from international partners including CDC and WHO, as we haveinitiated this rotavirus vaccine project, has enabled us to upgrademany of these aspects including GMP and GLP in the preparationof appropriate cell substrates and techniques, safety and toxicitytesting of the oral polio vaccines currently prepared at POLYVAC.

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[characterization of VP6 genes of human rotavirus isolates: correlation ofgenogroups with subgroups and evidence of independent segregation. J Virol

138 L.T. Le et al. / Vaccin

learly there are many hurdles ahead in the science, regulatoryssues, and clinical testing of these candidates, but the govern-

ent’s commitment to this internationally collaborative projectas helped us think more deeply about how to approach a problemhat affects infants throughout the world.

cknowledgments

We thank Dang Mai Dung, Nguyen Thi Quy, Nguyen Cong Long,nd Bui Huy Phuong for technical assistance and Wendi Kuhnert forritical reading of the manuscript. Technical support was providedy the Gastroenteritis and Respiratory Viruses Laboratory Branch,ivision of Viral Diseases and Biologics Branch, Division of Scientificesources, the Centers for Disease Control and Prevention, Atlanta,eorgia, USA.

onflict of interest

None.

unding sources

Supported by grants form the Ministry of Health and the Min-stry of Science and Technology, Vietnam, and the World Healthrganisation.

The findings and conclusions in this report are those of theuthors and do not necessarily represent the views of U.S. CDC.

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