Manu Tuilagi, Ben Youngs and Joe Marler have all withdrawn from the England training squad.
Worcester's Nick Schonert was also unable to join up with the squad in Bristol on Sunday night.
Marler withdrew on Sunday afternoon due to personal reasons, with Exeter's Ben Moon - capped by England at U16, U18 and U20 level - called up as a replacement.
Tuilagi, Youngs and Schonert all pulled out due to injury on Sunday evening.
Leicester Tigers centre Tuilagi, who last played for England during the 2016 Six Nations, had been named in the training squad for the first time in over a year.
But he has now been replaced by club team-mate Jordan Olowofela, who helped England reach the World Rugby U20 Championship final this summer
England's training cap runs until Tuesday, while a squad will be named on October 18 for the Quilter Internationals in November before they fly to Portugal for some final preparations.
England will play South Africa (November 3), New Zealand (November 10), Japan (November 17) and Australia (November 24) at Twickenham Stadium with 3pm kick-offs, live on Sky Sports.
Updated England training squad
Forwards (19): Tom Curry (Sale Sharks), Jamie George (Saracens), Dylan Hartley (Northampton Saints), Nathan Hughes (Wasps), Nick Isiekwe (Saracens), Maro Itoje (Saracens), George Kruis (Saracens), Joe Launchbury (Wasps), Courtney Lawes (Northampton Saints), Zach Mercer (Bath Rugby), Ben Moon (Exeter Chiefs), Michael Rhodes (Saracens), Chris Robshaw (Harlequins), Brad Shields (Wasps), Kyle Sinckler (Harlequins), Billy Vunipola (Saracens), Mako Vunipola (Saracens), Harry Williams (Exeter Chiefs), Mark Wilson (Newcastle Falcons).
Backs (15): Chris Ashton (Sale Sharks), Mike Brown (Harlequins), Danny Care (Harlequins), Joe Cokanasiga (Bath Rugby), Elliot Daly (Wasps), Nathan Earle (Harlequins), Owen Farrell (Saracens), George Ford (Leicester Tigers), Alex Lozowski (Saracens), Jonny May (Leicester Tigers), Jack Nowell (Exeter Chiefs), Dan Robson (Wasps), Henry Slade (Exeter Chiefs), Ben Te'o (Worcester Warriors), Jordan Olowofela(Leicester Tigers).